Combined Molecular and Elemental Mass Spectrometry Approaches
for Absolute Quantification of Proteomes: Application to the
Venomics Characterization of the Two Species of Desert Black
Cobras, Walterinnesia aegyptia and Walterinnesia morgani
Juan J. Calvete,*Davinia Pla, Johannes Els, Salvador Carranza, Maik Damm, Benjamin-Florian Hempel,
Elisa B. O. John, Daniel Petras, Paul Heiss, Ayse Nalbantsoy, Bayram Göcmen, Roderich D. Sussmuth,
Francisco Calderón-Celis, Alicia Jiménez Nosti, and Jorge Ruiz Encinar
Cite This: J. Proteome Res. 2021, 20, 5064−5078
Read Online
ACCESS Metrics & More Article Recommendations *
sıSupporting Information
ABSTRACT: We report a novel hybrid, molecular and elemental mass spectrometry (MS) setup for the absolute quantification of
snake venom proteomes shown here for two desert black cobra species within the genus Walterinnesia,Walterinnesia aegyptia and
Walterinnesia morgani. The experimental design includes the decomplexation of the venom samples by reverse-phase
chromatography independently coupled to four mass spectrometry systems: the combined bottom-up and top-down molecular
MS for protein identification and a parallel reverse-phase microbore high-performance liquid chromatograph (RP-μHPLC) on-line
to inductively coupled plasma (ICP-MS/MS) elemental mass spectrometry and electrospray ionization quadrupole time-of-flight
mass spectrometry (ESI-QToF MS). This allows to continuously record the absolute sulfur concentration throughout the
chromatogram and assign it to the parent venom proteins separated in the RP-μHPLC-ESI-QToF parallel run via mass profiling.
The results provide a locus-resolved and quantitative insight into the three desert black cobra venom proteome samples. They also
validate the units of measure of our snake venomics strategy for the relative quantification of snake venom proteomes as % of total
venom peptide bonds as a proxy for the % by weight of the venom toxins/toxin families. In a more general context, our work may
pave the way for broader applications of hybrid elemental/molecular MS setups in diverse areas of proteomics.
KEYWORDS: snake venomics, combined top-down and bottom-up venomics, hybrid elemental and molecular mass spectrometry,
absolute quantification of venom proteome, desert black cobra, Walterinnesia aegyptia, Walterinnesia morgani
1. BIOLOGICAL SIGNIFICANCE
The development of quantitative protocols during the first
decade of the 21st century has represented a major advance in
the field of proteomics. However, given the inherently
nonquantitative nature of molecular mass spectrometry (MS),
the absolute quantification of the proteome components is still a
challenge. Absolute quantification through molecular MS
requires spiking the experimental sample with a certified
Received: July 23, 2021
Published: October 4, 2021
Articlepubs.acs.org/jpr
© 2021 American Chemical Society 5064
https://doi.org/10.1021/acs.jproteome.1c00608
J. Proteome Res. 2021, 20, 5064−5078
Downloaded via TU BERLIN on September 15, 2023 at 10:53:36 (UTC).
See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
concentration of an isotopically labeled version for each target
molecule. To address the mismatch between the limited
quantification capabilities of molecular MS platforms and the
requirements of modern venomics, we have incorporated
inductively coupled plasma (ICP)-MS, a well-known technique
in the field of bioinorganic elemental analysis, into a novel
hybrid, molecular and elemental liquid chromatography−mass
spectrometry (LC−MS) workflow. This combines the unpar-
alleled molecular resolution of top-down MS and the absolute
quantification of sulfur (S) by ICP-MS as a proxy for the
absolute quantification of venom proteomes using a generic
sulfur standard. As a proof-of-concept, we have applied this
novel strategy to quantify three venom proteomes of the two
desert black cobra species within the genus Walterinnesia.
2. INTRODUCTION
Venoms and their associated venom-delivery systems are
intrinsically ecological traits that have evolved independently
in a wide range of lineages across all major phyla of the animal
tree of life.
1,2
Every ecosystem of our planet where there is
competition for resources harbors animals possessing toxic
weaponry. Venoms should therefore be understood as adaptive
responses shaped by natural selection in the context of reciprocal
selective predator−prey pressures that maximize the venomous
organisms’fitness in local environments through optimization of
the foraging/risk-of-predation balance and/or the self-defense
from predators.
3−9
Sixty to 50 million years ago, in the wake of the Cretaceous−
Paleogene boundary mass extinction event that ended the reign
of nonavian dinosaurs,
10,11
the emergence of venom represented
a key evolutionary innovation underpinning caenophidian snake
radiation.
12,13
Extant snake venoms are integrated phenotypes
comprised of mixtures of dozens to hundreds of peptides and
proteins, collectively referred to as toxins, which, despite
belonging to a limited number (2 < n< 20) of protein families,
possess a wide range of potent and specific pharmacological
activities capable to wreak havoc on the vital systems of the
animal prey or human victim.
14−18
Snakebite envenoming is an occupational hazard and a disease
of poverty that annually claims over 100 000 human lives
worldwide, particularly in the tropical and subtropical African
and Asian rural regions where ecological interactions between
venomous snakes and local people engaged in rural activities are
frequent.
19−22
Snakebite envenoming represents a multifactorial
One Health challenge.
20
Integration and contextualization of
conceptual frameworks from ecological venomics and clinical
toxinology can be mutually enlightening if snakebite envenom-
ing is analyzed from an ecological stance.
23
Hence, identifying
the specific pressures that tailored the composition and
bioactivitiesofvenomsacrosssnakecladesmayhave
implications for the clinical treatment of human envenom-
ings.
24−27
Abundance and toxicity are conjugated parameters of the
reference frame that explain the individual or synergistic
pharmacological profile of venom toxins. Since the turn of the
21st century, knowledge gathered from applications of omics
technologies, particularly the combination of next-generation
transcriptomics and mass spectrometry (MS)-based proteomics
platforms, has yielded compositional insights into snake venoms
from 238+ nominal species, mostly within the families Viperidae
(340 species of true vipers and pit vipers) and Elapidae (360
species of cobras, kraits, mambas, and sea snakes).
23,28,29
However, in contrast to the unprecedented highly resolved
descriptive knowledge of the compositional diversity of venoms,
estimating the abundance of their individual toxins has not
followed a parallel advance, e.g., toward absolute quantifica-
tion.
30−32
This is because MS is not an inherently quantitative
technique. A number of confounding factors may contribute to
the quantification of peptide ions in a mass spectrometer. Hence,
different analytes in any given sample may have different and
unpredictable ionization potentials, and the detection efficien-
cies for different m/zsignals are unequal.
33,34
Absolute
quantification through molecular MS requires, for each target
biomolecule, spiking the experimental sample with a corre-
sponding “Protein Standard for Absolute Quantification”
(PSAQ). PSAQs are whole synthetic isotopically labeled
analogues of the proteins to be quantified of certified
concentration and similar ionization efficiency as the target
analyte.
35,36
The recombinant or synthetic production of PSAQs
for each of the proteoforms of a venom proteome may be
technically possible, but it is not a feasible option in practice.
To address the mismatch between the quantification
capabilities of molecular MS-based proteomics platforms and
the requirements of modern venomics applications, we have
incorporated inductively coupled plasma (ICP)-MS, a well-
known technology in the field of bioinorganic elemental analysis,
into hybrid molecular and elemental LC−MS workflows for the
determination of sulfur (S) via isotope dilution analysis
(IDA)
37−39
as a proxy for the absolute quantification of
venom proteomes using a generic sulfur standard.
40,41
From
the absolute quantification of sulfur, the absolute amount of the
parental biomolecule can be calculated if the molar ratios of the
S-containing amino acids (cysteine and methionine) are known.
In this work, we have applied a recently developed strategy
where IDA has been replaced by the addition of C-containing
gas mixture (Ar/CO2) directly to the plasma to compensate for
changes in the organic composition of the mobile phase along
the reverse-phase (RP) chromatographic acetonitrile (ACN)
gradient.
42,43
This novel instrumental setup provides a stable
and corrected chromatographic signal, which is a simpler and
more easily automatable configuration than IDA and has
enhanced sensitivity compared to previous strategies. In this
work, we have applied this novel strategy to quantify the venom
proteomes of the two species of genus Walterinnesia.
The western species, Walterinnesia aegyptia Lataste 1887,
44
is
found in rocky and mountainous deserts, gravel and sandy
plains, and vegetated wadis in Egypt, border areas of Syria,
Jordan, Israel, and Palestine, while the eastern species,
Walterinnesia morgani (Mocquard, 1905), ranges from Syria,
Turkey, and northern Iraq, to Iran.
45,46
The situation in Saudi
Arabia is somewhat more confusing. Although Nilson and
Rastegar-Pouyani
46
draw a line separating the ranges of both
species based on some morphological characters, the fact that all
Saudi Arabian juveniles of Walterinnesia are black without the
typical narrow pinkish-brown cross-bands that characterize the
juvenile specimens of W. morgani from outside Saudi Arabia
(including the type locality in western Iran)
47
challenges Nilson
and Rastegar-Pouyani’s 2007 hypothesis
46
and suggests that all
Saudi Arabian specimens might belong to W. aegyptia.
48,49
Desert black snakes are medium-sized (maximum size of 1.3
m, average 0.8−1.2 m) strictly terrestrial snakes characterized by
a largely nocturnal and fossorial mode of life.
49
They are quick-
moving snakes that actively prey at night on dhub or spiny-tailed
lizards, toads, snakes, and occasionally birds and mice.
44,50
Desert black snakes usually bite their prey sideways at short
distances and often use constriction in addition to their potent
Journal of Proteome Research pubs.acs.org/jpr Article
https://doi.org/10.1021/acs.jproteome.1c00608
J. Proteome Res. 2021, 20, 5064−5078
5065
(intraperitoneally, i.p. LD50 of 0.175 mg/kg in 200−250 g adult
male albino rats) neurotoxic venom to kill the prey.
51
Desert
black snakes are reluctant to strike but will bite if cornered or
threatened. A few bite cases, although no recent fatalities, have
been documented. Bites may result in localized pain and
swelling, fever, generalized weakness, respiratory distress,
double vision, nausea, and vomiting.
50,52,53
The comprehensible
venomics characterization here reported may lay the ground-
work for future toxicovenomics analysis that defines the
functional map of the venoms of the black desert snakes.
3. MATERIALS AND METHODS
3.1. Venoms and Reagents
Venom samples of Saudi Arabian W. aegyptia [CN6136 (adult
female, Riyadh)], Egyptian Sinai Peninsula [CN6137 (adult
male), CN6138 (adult female), CN6139 (juvenile male), and
CN6140 (juvenile female)] were obtained, with permission and
under the supervision of the Environment and Protected Areas
Authority, Government of Sharjah (UAE), from the live
collection maintained at the Breeding Centre for Endangered
Arabian Wildlife. To clarify the taxonomy of the specimen from
Riyadh, Saudi Arabia, two mitochondrial (16S rRNA and
cytochrome oxidase I) and one nuclear (melanocortin 1
receptor) genes were PCR amplified and sequenced for
specimen CN6136 from Riyadh and specimen CN6137 from
the Sinai (see above) using the same primers and con-
ditions.
54,55
The sequences of the two specimens were nearly
identical, presenting one change in 530 base pairs (bp) of the
16S rRNA, one change in 669 bp of the cytochrome oxidase I;
and 0 changes in 686 bp of the melanocortin 1 receptor
(GenBank Accession numbers MZ520318−MZ520323). The
results of the comparison of the mitochondrial and nuclear DNA
data unambiguously identify the snake sample from Riyadh as
W. aegyptia and clearly show that the taxonomic hypothesis of
the genus Walterinnesia and especially the division between W.
aegyptia and W. morgani within Saudi Arabia by Nilson and
Rastegar-Pouyani
46
is incorrect and should be revised using
molecular data.
The venom of W. morgani was collected from one adult female
captured in November 2007 near Corten village at Kilis and
Gaziantep province boundaries
56
and maintained since in
captivity at the Reptile Biology and Ecology Research
Laboratory (Zoology Section, Department of Biology, Ege
University). Venom was obtained by allowing the snakes to bite
a paraffin-covered laboratory beaker without pressing the venom
glands. The venom sample was centrifuged at 4 °C at 2000gfor
10 min, and the supernatants were immediately lyophilized and
the samples stored at 4 °C. The Ege University Local Ethics
Committee (process number 2013-050) approved the exper-
imental protocol.
Inorganic sulfur ICP standard (1000 mg/L) was purchased
from SPEX CertiPrep, INC. (New Jersey). Solutions were
prepared in ultrapure (18.2 MΩ·cm) water. HPLC grade
acetonitrile (ACN) was purchased from Fischer Scientific, and
formic acid (FA) was purchased from Merck KGaA (Germany).
3.2. Determination of the Murine Median Lethal Dose
(LD50)ofW. morgani Venom
The murine median lethal dose (LD50) of pooled W. morgani
crude venom was determined through the up-and-down method
recommended by the Organization for Economic Cooperation
and Development (OECD) Guidelines (Test No. 425).
57,58
To
this end, increasing venom amounts (0.1, 1, and 5 mg of total
venom proteins per kg of mouse body weight) dissolved in 100
μL of physiological (0.9%) saline solution were administered
intraperitoneally (i.p.) to groups of five Balb/c mice. Control
mice received a single i.p. injection of sterile saline (0.9%, 100
μL). Deaths were recorded 24 h after venom injection, and the
LD50 was calculated through a nonlinear regression fitting
procedure in GraphPad Prism 5 (version 5.01).
3.3. Molecular Mass Spectrometric Characterization of the
Venom Arsenals of the Desert Black Snakes, W. aegyptia
and W. morgani
Initial reverse-phase chromatographic profiling of the five W.
aegyptia venom samples showed a conserved protein elution
pattern in the four Egyptian Sinai Peninsula specimens
[CN6137 (adult male), CN6138 (adult female), CN6139
(juvenile male), and CN6140 (juvenile female)], and a different
pattern for the venom of the adult female specimen from Riyadh
(CN6136). Venoms of this Saudi Arabian snake and the
Egyptian adult male specimen CN6137 were selected for
comparing their proteome toxin composition between them-
selves and with the venom proteome of the adult female W.
morgani (Corten village, Turkey) specimen.
3.3.1. Bottom-Up Decomplexation and Relative
Quantification of the W. aegyptia and W. morgani
Venom Proteomes. For reverse-phase chromatographic
decomplexation, 2 mg of crude lyophilized venom samples
was dissolved in 100 μL of 0.05% trifluoroacetic acid (TFA) and
5% acetonitrile, and the insoluble material was spun down in an
Eppendorf centrifuge at 13 000gfor 10 min at room temper-
ature. Decomplexation of the venom proteomes was performed
according to the reverse-phasehigh-performanceliquid
chromatography (RP-HPLC)/sodium dodecyl sulfate-polya-
crylamide gel electrophoresis (SDS-PAGE) protocol of our
“snake venomics”strategy
59,60
with minor modifications.
61
To
this end, 40 μL was applied to a RP-HPLC Teknokroma Europa
C18 (250 mm ×4 mm, 5 μm particle size, 300 Å pore size)
column. The venom proteins were fractionated using an Agilent
LC 1100 high-pressure gradient chromatography system
equipped with a diode array detector, applying a linear gradient
of 0.1% (v/v) TFA in water (solution A) and in 70% acetonitrile
(solution B): 0−5 min isocratically with 5% B (0.1% TFA in
ACN), followed by the following linear gradient steps: 5−25% B
(10 min), 25−45% B (60 min), and 45−70% (10 min), at 1 mL/
min. Protein peaks were recorded at λ= 215 nm, and the eluate
was manually collected and dried using a vacuum centrifuge
(SpeedVac, Thermo Savant).
Molecular masses of the RP-HPLC-separated venom proteins
were estimated by nonreducing and reducing SDS-PAGE (on
15% polyacrylamide gels) or determined by nano-Acquity
UltraPerformance LC (UPLC) equipped with a BEH130 C18
(100 μm×100 mm, 1.7 μm particle size) column in-line with a
Waters SYNAPT G2 high-definition mass spectrometer, as
previously described.
62
Protein bands of interest were excised from Coomassie
Brilliant Blue-stained SDS-PAGE gels and subjected to
automated in-gel reduction and alkylation using a Genomics
Solution ProGest Protein Digestion Workstation.
62
Tryptic
digests were submitted to MS/MS analysis using the same Mass
Spectrometry System and chromatographic separation con-
ditions as above. Doubly and triply charged ions were selected
for CID-MS/MS. Fragmentation spectra were submitted to the
MASCOT Server (version 2.6) at http://www.matrixscience.
com and matched against the last update of the NCBI
Journal of Proteome Research pubs.acs.org/jpr Article
https://doi.org/10.1021/acs.jproteome.1c00608
J. Proteome Res. 2021, 20, 5064−5078
5066
nonredundant database, including the W. aegyptia venom gland
transcriptomic data deposited with the SRA and TSA databases
of NCBI (BioProject accession number PRJA506018) publicly
available in the MassIVE repository under the accession number
MSV000081885 (ftp://massive.ucsd.edu/MSV000081885/)
and ProteomeXchange with the accession number
PXD008597. Good quality unmatched fragmentation spectra
were manually (de novo) sequenced, and the assigned peptide
sequences matched to homologous snake venom proteins
available in the NCBI nonredundant protein sequences database
using the default parameters of the BLASTP program (https://
blast.ncbi.nlm.nih.gov/Blast.cgi).
62
For the relative quantification of the venom arsenals of W.
aegyptia and W. morgani, we applied the three-step hierarchical
venom proteome quantification protocol developed in our
laboratory
60,63
to compile the relative composition of toxin
families in the venom proteome of W. aegyptia and W. morgani
venom samples. The calculated relative abundances correspond
to the % by weight (g/100 g) of the pure venom component.
41
3.3.2. Top-Down Venomic (TDV) Analysis of the W.
aegyptia and W. morgani Venom Weaponry. Denaturing
top-down proteomic experiments were performed as previously
described.
58,64−66
In short, 100 μg of crude venoms was
dissolved at a final concentration of 10 mg/mL in aqueous 1%
(v/v) formic acid (FA). Dissolved venom was centrifuged at
20 000gfor 5 min, and the supernatant was mixed with 30 μLof
citrate buffer (0.1 M, pH 3.0). For reduction of disulfide bonds,
10 μL of 0.5 M tris(2-carboxyethyl)phosphine (TCEP) was
added to one-half of the sample and incubated for 30 min at 65
°C. The other half of the sample was supplemented with 10 μL
of ultrapure water. The samples were centrifuged at 20 000gfor
5 min, and 10 μL of reduced and nonreduced samples was
analyzed each by LC (RP-HPLC)-high-resolution (HR)
electrospray ionization (ESI)-MS/MS. Technical duplicates
were performed by two different LC-ESI-HR-MS setups, W.
aegyptia venoms by setup (A) and W. morgani venom by setup
(B).
Setup (A) was performed in an LTQ Orbitrap XL mass
spectrometer (Thermo, Bremen, Germany) coupled to an
Agilent 1200 HPLC system equipped with a Supelco Discovery
300 Å C18 (2.1 mm ×150 mm, particle size, 3 mm) column.
The column was developed with a gradient of 0.1% FA in water
(solution A) and acetonitrile (ACN) (solution B) at a flow rate
of 0.3 mL/min. Chromatographic conditions and ESI settings
were as previously described.
65
Setup (B) LC−MS/MS experiments were done using a
Vanquish ultra-high-performance liquid chromatography
(UHPLC) system equipped with a 300 Å pore size, 2 mm ×
150 mm column size, 3 μm particle size Supelco Discovery BIO
wide C18 column thermostatted at 30 °C and hyphenated to a
Q-Exactive quadrupole orbital ion trap (Thermo Fisher
Scientific) as previously described.
66
MS/MS spectra were
obtained in the DDA mode at a mass resolution of 140 000 (at
m/z200), and the three most abundant ions of the survey scan
were selected for MS/MS.
3.3.2.1. Top-Down MS Analysis and Intact Mass Profiling.
Thermo data (.raw) were converted to a centroided mass
spectrometry data format (.mzXML) using the MSconvert
software of the ProteoWizard package (http://proteowizard.
sourceforge.net; version 3.0.10577)
67
with a peak picking level
of 1+. The. mzXML data were deconvoluted to a. msalign file
using TopFD (http://proteomics.informatics.iupui.edu/
software/toppic/; version 1.3) with a maximum charge of 50,
a maximum mass of 100 000 Da, an MS1 S/N ratio of 3.0, an
MS2 S/N ratio of 1.0, an m/zprecursor window of 3.0, and an
m/zerror of 0.02. The final sequence annotation was performed
with TopPIC (http://proteomics.informatics.iupui.edu/
software/toppic/; version 1.3),
68
with decoy database, 15 ppm
mass error tolerance, E-value cutoffat 0.01 by E-value
computation, 1.2 Da PrSM cluster error tolerance, and a
maximum of 2 mass shifts (±500 Da). Spectra were matched
against a W. aegyptia database as well as against a reviewed
Elapinae database (https://www.uniprot.org/, 518 entries,
20.12.2020), manually validated, and visualized using the MS
and MS/MS spectra using Qual Browser (Thermo Xcalibur 2.2
SP1.48) and Freestyle (Thermo Xcalibur 1.6.75.20). The
XTRACT algorithm of Thermo Xcalibur was used to
deconvolute isotopically resolved spectra.
3.4. Absolute Quantification of Sulfur by Capillary RP-HPLC
On-Line to Inductively Coupled Plasma (ICP-MS/MS)
Elemental Mass Spectrometry
Lyophilized venom samples were reconstituted in ultrapure
water to a final sample concentration of ∼0.5 mg/mL. The
venom proteins contained in 1 μL were separated by RP-HPLC
using a Sigma-Aldrich (Steinheim, Germany) 150 mm ×0.3 mm
C4 capHPLC column (BIOShell A400, 3.4 μm particle size, 400
Å pore size) run on an Agilent Technologies (Waldbronn,
Germany) Infinite Capillary HPLC 1260 Series system
equipped with an autosampler module and a Spark Holland
oven heating system (Mistral, the Netherlands). The column
was developed at 80 °Cataflow rate of 4.5 mL/min with a
gradient of 0.2% FA in water (solution A) and 0.2% FA in
acetonitrile (solution B). Optimized chromatographic con-
ditions (min % B) were as follows: W. aegyptia [CN6137 (adult
male, Sinai Peninsula, Egypt)] 0−2, 2−2, 4−8, 11−15, 13−16,
17−16, 27−18, 39−22, 57−35, 67−60, and 73−90; W. aegyptia
[CN6136 (adult female, Riyadh, Saudi Arabia)] 0−1.5, 5−2, 6−
10.8, 13−11.1, 15−24.1, 27−24.2, 28−29.2, 35−29.5, 38−65,
43−75, and 45−90; W. morgani (adult female, Corten village,
Turkey) 0−1.5, 5−1.5, 8−10, 18−15, 30−25, 45−30, 55−70,
and 60−90. Complete protein recovery from the chromato-
graphic column, an essential requisite to accurately quantify
venom proteins with ICP-MS, was assessed by injecting in
triplicate the sample under flow injection analysis (FIA) prior to
the chromatographic analysis.
40
Capillary RP-HPLC FIA
conditions were the same as the starting conditions for venom
decomplexation, and both RP-HPLC fractionation and FIA
analysis used the same sample injection volume so that sulfur
mass balance could be directly determined.
For the absolute quantification of the venom components,
sulfur was continuously quantified through ICP-MS/MS
analysis.
69
The analytical potential of sulfur measurement for
the general quantitative analysis of cysteine and/or methionine-
containing proteins and peptides
70
has already been validated
for venom proteome quantification.
40
The ICP-MS/MS system
used was an Agilent 8900 triple quadrupole ICPQQQ-MS
(Tokyo, Japan). The sulfur quantification standard was injected
using capFIA prior to the capHPLC analysis.
43
This standard
can be any compound of certified concentration that contains
sulfur because of the species-independency of the elemental
response in the detection. External calibration provided the
sulfur response factor (i.e., the peak area of S per unit of
concentration of the S standard injected) and was applied using
eq 1 to quantify the sulfur present in each chromatographic peak
of the samples
Journal of Proteome Research pubs.acs.org/jpr Article
https://doi.org/10.1021/acs.jproteome.1c00608
J. Proteome Res. 2021, 20, 5064−5078
5067
=·CC(area /area )
S
peak S
standard S
peak S
standard (1)
Absolute protein quantification by ICP-MS requires maintain-
ing the elemental response factor constant along the complete
chromatographic analysis. To fulfill this requirement, a total
consumption nebulizer (capillary LC interface, Agilent) was
used between the capHPLC and ICP-MS/MS systems.
71
For
signal variation correction (<6% relative standard deviation) and
enhanced sensitivity,
72
continuous addition of 50 mL/min
carbon dioxide (CO2/Ar, 10:90) gas mixture (Air liquide,
Madrid, Spain) to the ICP-MS plasma was controlled with a
Bronkhorst Mass Flow Meter (the Netherlands). CO2/Ar was
mixed on-line with optional gas O2/Ar (20:80) (Air liquide,
Madrid, Spain) through a T-connection located between the exit
of the ICP-MS optional gas and the optional inlet of the
nebulization chamber.
3.4.1. Correlation between the Sulfur and the Protein
Chromatographic Profiles. Knowledge of the stoichiometry
of sulfur atoms in a protein sequence is needed to transform
sulfur concentration into protein concentration. For this
purpose, the identity of the toxins eluted along the chromato-
graphic separation of venom was achieved through parallel ESI-
MS native mass profiling in the same chromatographic peaks
analyzed by ICP-MS/MS. ESI-MS mass profiling was recorded
with a Bruker Daltonics (Bremen, Germany) ESI-QToF MS
Impact II instrument. Protein identification was inferred
through a comparison of the masses of Walterinnesia venom
proteins assigned by bottom-up and top-down proteomics
analyses (Supporting Information Tables S2−S5) with those
gathered through venom gland transcriptomic-assisted top-
down analysis of a W. aegyptia venom sample (Supporting
Information Table S1) deposited in NCBI SRA and TSA
databases associated with BioProject PRJA506018. LC−MS/
MS.raw and centroid.mzXML data are publicly available in the
MassIVE repository under the accession number
MSV000081885 (ftp://massive.ucsd.edu/MSV000081885)
and ProteomeXchange (accession number PXD008597).
73
4. RESULTS AND DISCUSSION
4.1. Experimental Design
We report the application of a novel MS-based workflow for the
absolute quantification of the locus-resolved venom proteomes
of two species of desert black cobras, W. aegyptia and W.
morgani. The experimental setting, schematized in Figure 1,
includes decomplexation of the venom samples by reverse-phase
chromatography independently coupled to each of four mass
spectrometry systems. Protein identification was accomplished
through a combination of bottom-up (Figure 1,2a−c) and top-
down (Figure 1, 3) molecular MS-based workflows. Bottom-up
venomics (BUV) relies on in-gel tryptic digestion of SDS-PAGE
bands of the venom proteins separated using RP-HPLC (Figure
1, 2a), ESI-MS/MS sequencing of the resulting tryptic peptides
(Figure 1, 2b), and matching the recorded product ion spectra
against a protein database with a search algorithm.
32,59,63
BUV
takes advantage of venom fractionation to simultaneously
quantitate the relative abundances of the different venom
components (Figure 1, 2c). On the other hand, in the top-down
venomics (TDV) approach (Figure 1,3a−c) front-end-
fractionated disulfide-bond-reduced intact polypeptide ions
generated by electrospray ESI are manipulated and dissociated
inside a high-resolution Fourier transform ion-trapping (e.g.,
Figure 1. Schematic of the combined molecular and elemental mass spectrometry methodology applied in this work for the absolute quantification of
the venom proteomes of black desert cobras, W. aegyptia and W. morgani. The workflow comprises four RP-HPLC venom protein separations and
downstream analysis through bottom-up (2a−c) and top-down (3a−c) venomics and combined parallel mass profiling (4a,b and 6) and absolute
sulfur determination by ICPQQQ-MS/MS (5a−e). Continuous sulfur quantification along the chromatographic run was correlated with the
molecular masses measured in the parallel RP-capHPLC-ESI-QToF run for the parent venom toxins (6) and assigned to amino acid sequences
gathered from bottom-up and top-down venomics (7). Molar ratios sulfur/protein [μmol S/n(Cys + Met)] computed throughout the chromatogram
were translated into the corresponding absolute protein amounts (8) using the equation [μmol S/n(Cys + Met)] ×MTi =μg Ti, where n(Cys + Met) is
the number (n) of cysteine and methionine residues in the amino acid sequence of toxin “i”(Ti) and MTi is the ESI-MS determined monoisotopic
molecular mass of toxin i.
Journal of Proteome Research pubs.acs.org/jpr Article
https://doi.org/10.1021/acs.jproteome.1c00608
J. Proteome Res. 2021, 20, 5064−5078
5068
orbitrap) mass spectrometer (Figure 1, 3a).
64,65,74
A benefitof
TDV is that the intact mass of every proteoform is retained,
overcoming the challenge of BUV regarding the characterization
of small proteins that often yield an insufficient number of
proteolytic peptides for unequivocal proteoform identification.
Locus-resolved toxin identification by top-down MS/MS
analysis (Figure 1, 3b) complements BUV and represents an
important progression toward a full qualitative description of a
venom’s proteome (Figure 1, 3c). However, molecular mass
spectrometry used in BUV and TDV is inherently not a
quantitative technique, and the proposed absolute protein
quantification strategies are limited by the need for proteotypic
internal standards for each target protein.
32,42,75
To overcome
this limitation, our workflow includes a hybrid elemental and
molecular MS configuration where two identical venom samples
are submitted to decomplexation through parallel RP-μHPLC
run under identical chromatographic conditions. The identity of
the toxins along the chromatographic separation was inferred
through ESI-QToF mass profiling (Figure 1, 4a) matching the
monoisotopic molecular masses calculated for mature toxins
recorded in the BUV and TDV analyses or calculated from a
homologous venom gland transcriptomic database
73
(Support-
ing Information Table S1)(Figure 1, 4b). For the absolute
quantification of the venom’s proteins, sulfur concentration was
continuously measured throughout the chromatogram via ICP-
MS/MS (Figure 1, 5a). Then, the sulfur response factor
obtained from a certified S-containing generic compound
(Figure 1, 5b) injected using capFIA prior to the capHPLC
analysis (i.e., the peak area of S per unit of concentration of the S
standard injected) was used to translate the individual peak areas
of the different peaks into sulfur concentration (Figure 1, 5d,e).
Compared to previous ICP quantification approaches using
online isotope dilution analysis (IDA) to keep both the protein
response factor and the isotopic tracer added continuously
constant along the whole chromatogram, the recently
introduced strategy of continuous addition of 50 mL/min
carbon dioxide (CO2/Ar, 10:90) gas mixture to the plasma
provides excellent signal variation corrections along the
chromatographic separation for all elements simultaneously
(<6 RSD%) while maintaining sensitivity enhancement (2−9-
fold).
43,71
This approach makes the use of isotopic dilution
analysis unnecessary, thereby simplifying the mathematical
treatment of the data (Figure 1, 5c,d). Sulfur quantified along
the chromatographic run was assigned to the parent venom
proteins separated in the parallel RP-capHPLC-ESI-QToF run
(Figure 1, 6), and the stoichiometry S/P [mol S (Cys + Met)/
mol Protein] was computed throughout the chromatogram from
the amino acid sequences (Figure 1, 7) and translated into the
corresponding absolute protein amounts (Figure 1, 8).
4.2. Combined Bottom-Up and Top-Down MS
Characterization and Relative Quantification of W. aegyptia
and W. morgani Venom Proteomes
Combined bottom-up and top-down MS approaches were
applied to match the RP-HPLC-separated venom profiles of two
W. aegyptia specimens (Sinai Peninsula, Egypt, and Riyadh,
Saudi Arabia) and a venom sample from a W. morgani specimen
originating from Corten village (Turkey) to a W. aegyptia venom
gland transcriptomic database. Figures 2 and 3display RP-
HPLC decomplexation of the venom proteomes of the Egyptian
and Saudi Arabian W. aegyptia (panels A and B, respectively)
and Turkish W. morgani (panel C). Figure 2 displays the
bottom-up venomics analysis of these three desert black cobras’
Figure 2. Bottom-up venomics analysis of the toxin arsenal of desert
black cobras, W. aegyptia and W. morgani. Panels (A−C) display,
respectively, reverse-phase chromatographic separations of the venom
proteins of two W. aegyptia specimens (Sinai Peninsula, Egypt, and
Riyadh, Saudi Arabia) and a venom sample from a W. morgani specimen
original from Corten village (Turkey). For venomics analyses,
chromatographic fractions were collected manually and analyzed by
SDS-PAGE (inset) under nonreduced (upper panels) and reduced
(lower panels) conditions. Protein bands were excised, in-gel digested
with trypsin, and the resulting proteolytic peptides were fragmented
through LC-nESI-MS/MS. Parent proteins were identified by database
searching (against the last update of the NCBI nonredundant database,
including the W. aegyptia venom gland transcriptomic data deposited
with the SRA and TSA databases, Supporting Information Table S1)
and de novo sequencing followed by BLAST analysis (Supporting
Information Tables S2−S4). Picture of W. aegyptia specimens displayed
in panels (A) and (B) were taken by Salvador Carranza. Picture of W.
morgani, Bayram Gocmen.
Journal of Proteome Research pubs.acs.org/jpr Article
https://doi.org/10.1021/acs.jproteome.1c00608
J. Proteome Res. 2021, 20, 5064−5078
5069
venom proteomes applying primary RP-HPLC snake venom
protein separations with eluate detection at the peptide bond
absorbance wavelength (215 nm) and secondary subfractiona-
tion of the chromatographic peaks by SDS-PAGE. Figure 3
displays the total ion current (TIC) profiles of the same venom
samples analyzed in Figure 2. The simple comparison of the
separations of the same proteomes visualized by quantifying
different parameters clearly shows large differences between the
relative abundances of the venom components as a function of
the monitored parameter, absorbance vs TIC. The reason for
this discrepancy has to be attributed to the different physical
principles underlying the techniques used to monitor similar RP-
HPLC eluates. Monitoring the reverse-phase column eluate at
the absorbance wavelength of the peptide bond provides a
measure of the concentration of peptide bonds along the
chromatographically separated fractions. The relative abundan-
ces of a venom toxin arsenal estimated as the ratio of the peak
area to the total area of the venom proteins in the reverse-phase
chromatogram have a unit of “% of the total chromatographic
peptide bond concentration,”which conceptually is a proxy of
the weight % “g toxini/100 g of total venom proteins.”
41
On the
other hand, the TIC chromatograms recorded through TDV
(Figure 3) represent the summed dimensional intensity across
the entire range of masses detected at every point of the RP-
HPLC chromatogram. Different ionization efficiency/detect-
ability intrinsic to polypeptide ions limit the applicability of TIC
to estimate relative protein abundances.
76,77
Hence, top-down
MS data were used here only for the purpose of complementing
and expanding the bottom-up qualitative identification of the
different proteins/proteoforms present in the three desert black
cobra venom proteomes sampled (Supporting Information
Tables S1−S5).
Figure 4 displays a comparison of the relative abundances of
the toxin families comprising the venom proteomes of the W.
aegyptia and W. morgani venom proteomes quantified by BUV,
and the identity of the major toxin family members was gathered
Figure 3. Total ion current (TIC) profiles of reduced venom proteins of Egyptian and Saudi Arabian W. aegyptia (panels A and B, respectively) and
Turkish W. morgani (panel C) separated by reverse-phase HPLC. Peak numbering same as in the homologous UV-monitored chromatographic traces
displayed in Figure 2. Top-down MS identifications of proteins in the proteomes of W. aegyptia and W. morgani venoms are listed in the Supporting
Information Table S5 and integrated with the homologous bottom-up datasets in the Supporting Information Tables S2−S4.
Journal of Proteome Research pubs.acs.org/jpr Article
https://doi.org/10.1021/acs.jproteome.1c00608
J. Proteome Res. 2021, 20, 5064−5078
5070
by TDV (Supporting Information Tables S2−S4). The three
venoms are made up of four dominant toxin families, three-
finger toxin (3FTx, 24−51%), phospholipase A2(PLA2,19−
41%), cysteine-rich secretory protein (CRISP, 8−16%), and
Kunitz-type serine proteinase inhibitor-like protein (KUN, 7−
13%). Two other toxin families, snake venom metalloprotei-
nases of class PIII (PIII-SVMP) and L-amino acid oxidase
(LAO), are present in medium abundances (6−7%) in the
venom proteomes of W. aegyptia from Sinai Peninsula (Egypt)
and W. morgani, and the three venoms contain a set of 3−6 low-
abundance (<0.1%) proteins, including 5′-nucleotidase (5′NT),
endonuclease (Endo), phosphodiesterase (PDE), nerve and
vascular endothelial growth factors (NGF and VEGF), and
acetylcholinesterase (AcChol) (Figure 4; Supporting Informa-
tion Tables S2−S5). The small set of individual major toxins that
make up the venom protein families, 3FTx (2−4 proteins),
KUN (3−4 proteins), and PLA2(two highly homologous
molecules), is highly conserved among the three Walterinnesia
venom proteomes, but their relative abundances vary (Figure 4).
Although lethal doses for the individual Walterinnesia venom
toxins have not been reported, making an informed discussion
on the impact of compositional variability on the overall toxicity
of the venoms impossible, the i.p. murine LD50 of W. morgani
venom 0.66 (CI95 % 0.13−3.37) μg/g mouse body weight (this
work) is comparable to the i.v. LD50 reported for W. aegyptia
(0.79 (0.62−1.09) μg/g mouse).
73
Figure 4. Pie charts displaying the BUV quantified relative occurrence (in the percentage of total venom proteins) of the different protein families in
the venom proteome of desert black cobras, W. aegyptia (Sinai Peninsula, Egypt) (panel A), W. aegyptia (Riyadh, Saudi Arabia) (panel B), and W.
morgani (Corten village, Turkey) (panel C). Major TDV-identified family member components are highlighted in each pie chart. Acronyms: 3FTx,
three-finger toxin; KUN, Kunitz-type serine proteinase inhibitor-like protein; CRISP, cysteine-rich secretory protein; PLA2, phospholipase A2;5′NT,
5′nucleotidase; svNGF, snake venom nerve growth factor; PIII-SVMP, snake venom metalloproteinase of class PIII; PDE, phosphodiesterase; LAO, L-
amino acid oxidase; VEGF, vascular endothelial growth factor; AcChol, acetylcholinesterase; Endo, endonucleotidase.
Journal of Proteome Research pubs.acs.org/jpr Article
https://doi.org/10.1021/acs.jproteome.1c00608
J. Proteome Res. 2021, 20, 5064−5078
5071
4.3. Absolute Venom Protein Quantification via a Hybrid
Elemental and Molecular Mass Spectrometry Configuration
The development during the first decade of the 21st century
33
of
quantitative MS-based strategies represented a major advance in
the proteomics arena. An inherent drawback of absolute protein
quantification based on molecular MS approaches is the
requirement of stable isotope-labeled analogous standards for
each target molecule.
37,78
In this context, since its introduction
in the early 2000s,
79
heteroatom-tagged elemental MS is gaining
momentum as a versatile technique for the absolute
quantification of biomolecules without specific standards due
to its capability to quantify heteroatoms (any element except C,
H, N, O, and F) present in the structure of the target
biomolecules.
72,80,81
Leveraging on a new approach for
removing polyatomic interference using a triple quadrupole
inductively coupled plasma (ICP) mass spectrometry config-
uration,
69
we have recently developed a hybrid elemental and
molecular MS platform based on a reverse-phase (RP) capillary
μHPLC hyphenated to an ICP-MS/MS mass spectrometer and
on-line IDA for the absolute quantification of the venom
proteomes of the Mozambique spitting cobra (Naja mossamb-
ica), the black-necked spitting cobra (Naja nigricollis), the New
Guinea small-eyed snake (Micropechis ikaheka), and the Papuan
black snake (Pseudechis papuanus).
40,41
In this approach, the
combination of spiking a generic S-containing internal standard
to the sample and postcolumn addition of 34S provided the basis
for the absolute quantification of the RP-HPLC-separated S-
containing venom toxins, the identity of which was accom-
plished by ESI-QToF mass profiling along a parallel RP-HPLC
run.
32,71,72
Now, we have applied a newly developed protocol
where the addition of 50 mL/min carbon dioxide (CO2/Ar,
10:90) gas mixture directly to the plasma abolishes the need for
correcting sulfur response factor variation (>6%) along the
chromatographic separation using complex 34S-isotope dilution
procedures.
42,43
Our current workflow retains the hybrid elemental and
molecular MS configuration (Figure 1, 4a and 5a) of its
predecessor platform (Figure 1 of Calderon-Celis et al.).
41
Complete chromatographic column protein recovery is a strictly
necessary condition to achieve accurate ICP-MS-based generic
absolute protein quantification. Sample recoveries from the C4
capHPLC column, evaluated via Flow Injection Analysis, were,
respectively, 95 ±3, 92 ±1, and 102 ±1% for W. aegyptia (Sinai
Peninsula), W. aegyptia (Riyadh), and W. morgani (Corten
Table 1. Relative Quantification through Bottom-Up Venomics and Absolute Quantification via ICP-MS/MS of the Major and
Some Minor Components of the Venom Proteomes of the Desert Black Cobras, W. aegyptia (Sinai Peninsula, Egypt), W. aegyptia
(Riyadh, Saudi Arabia), and W. morgani (Corten village, Turkey)
a
ICP-MS/MS [mg/100 mg V] BUV [% venom proteome] mean ±SD ESI-QToF [% cps]
W.aegyptia (Sinai) 3FTX 22.6 24.4 23.5 ±0.9 65.8
KUN 13.8 13.1 13.5 ±0.4 28.5
PLA240.6 40.1 40.3 ±0.3 3.8
CRISP 9.8 8.1 8.9 ±0.8 0.6
svNGF 0.5 0.2 0.3 ±0.1 0.2
PIII-SVMP 12.6 6.6 9.6 ±2.9 1.1
LAO 6.7
5′NT 0.1
PDE 0.7
W. aegyptia (Riyadh)
3FTX 54.8 51.4 53.1 ±1.7 61.0
KUN 16.0 9.4 12.7 ±3.3 23.1
PLA212.6 18.8 15.7 ±3.1 14.9
CRISP 16.0 16.4 16.5 ±0.1 1.0
PIII-SVMP 1.3
LAO 2.23
VEGF 0.16
PDE 0.06
AcCHOL 0.2
Endo 0.01
W. morgani
3FTX 45.4 39.4 42.4 ±3.0 65.4
KUN 13.7 7.2 10.5 ±3.2 10.7
PLA227.0 26.1 26.6 ±0.5 21.4
CRISP 13.8 13.5 13.7 ±0.2 1.9
svNGF 0.001 0.010 0.0056 ±0.0044 0.7
PIII-SVMP 7.1
LAO 6.4
Endo 0.01
VEGF 0.16
5′NT 0.01
PDE 0.06
AcCh 0.7
a
For comparison, the relative abundances calculated from the mass signal intensity recorded in the ESI-QTof mass profiling analysis (cps, counts
per second) is also included.
Journal of Proteome Research pubs.acs.org/jpr Article
https://doi.org/10.1021/acs.jproteome.1c00608
J. Proteome Res. 2021, 20, 5064−5078
5072
village). ICP-MS sulfur quantification along the chromato-
graphic run was assigned to the parent venom proteins separated
in the parallel RP-capHPLC-ESI-QToF run (Figure 1, step 6)
and was then translated into the corresponding absolute protein
amounts (e.g., mg, μmoles) (Figure 1, step 8; Supporting
Information Tables S6−S8) using the stoichiometry mol S (Cys
+ Met)/mol Protein computed throughout the chromatogram
from the amino acid sequences, gathered from the bottom-up
and top-down venomics analyses. Table 1 compares the absolute
ICP-MS/MS quantifications of the major toxins of W. aegyptia
(Sinai Peninsula), W. aegyptia (Riyadh), and W. morgani
(Corten village) venoms, expressed as mg toxin/100 mg
venom, with the respective relative quantifications gathered
through our three-step bottom-up venom proteome quantifica-
Figure 5. Overlay of the ICP-MS mass flow chromatograms (red) and the ESI-MS chromatograms (black) of the venoms of (A) W. aegyptia (Sinai
Peninsula, Egypt), (B) W. aegyptia (Riyadh, Saudi Arabia), and (C) W. morgani (Corten village, Turkey). Peak matching displayed in the Supporting
Information Tables S6−S8 enabled correlating molecular peak identity and elemental S quantitation.
Journal of Proteome Research pubs.acs.org/jpr Article
https://doi.org/10.1021/acs.jproteome.1c00608
J. Proteome Res. 2021, 20, 5064−5078
5073
tion protocol (Materials and Methods Section 3.3.1).
60,63
The
reasonably good agreement between the values obtained by
ICP-MS/MS and bottom-up venomics for major toxin families
(Table 1) corroborated our previous assumption
41
that the “%
of the total venom proteome’s peptide bonds”represents a proxy
for the weight % (g/100 g) for the venom components and thus
has units of mg toxin/toxin family per 100 mg of total venom
proteins. Of course, this strategy is still prone to error, given that
the contribution to the molar absorption coefficient (ε) of each
protein species is not solely determined by its peptidebonds, but
the contribution of several amino acid residues must be taken
into account as well.
82
This miscalculation does not occur in ICP
quantification because the signal is directly proportional to the
concentration of sulfur. The relative abundances calculated from
the mass signal intensity (cps, counts per second, the number of
ions that hit the detector per unit of time) recorded in the ESI-
QToF mass profiling analysis (Figure 1, step 6) did not show a
consistent correlation with the ICP-MS/MS data (Table 1). No
toxin/family of toxins-associated pattern emerges from the data
displayed in Table 1 that would allow rationalizing and thus
eventually correcting the observed biases. On the other hand, for
all pairwise comparisons of homologous data obtained by ICP-
MS and by our bottom-up snake venomics approach,
32,60,63
the
standard deviation of the averaged value was within the range of
0.1−3.3% (Table 1).
5. CONCLUDING REMARKS AND PERSPECTIVES
Established in the 1990s as a powerful analytical technique,
molecular mass spectrometry has opened new experimental
approaches to address biological questions. However, molecular
mass spectrometry is not inherently quantitative, and this
analytical deficiency motivated the development of label-free
and isotopic labeling methods to determine the relative and
absolute abundance of biomolecules in complex biological
samples. ICP-MS, a type of elemental mass spectrometry
introduced in 1980
83
and available commercially soon after
1983, is a powerful analytical tool for trace elemental speciation
analysis of metals, semimetals, and several nonmetals (and their
different isotopes) at concentrations as low as ppq, one part per
quadrillion (1015).
69,84
More recently,
36
ICP-MS has emerged as
an alternative to overcome the absolute quantification
limitations of molecular MS. Implementation of ICP-MS in
the proteomics arena has been delayed by the fact that this
technique atomizes the sample and detects individual ionized
atomic elements. Therefore, the “elemental”information
yielded by ICP-MS cannot per se be used to differentiate the
different S-donor molecules of a mixture. Notwithstanding its
lack of molecular resolution, the omnipresence of sulfur in
proteins, together with the fact that proteins can be more and
more extensively and efficiently separated nowadays, i.e., by
advanced RP-HPLC, make the absolute protein quantification
via sulfur determination by ICP-MS a feasible strategy. A major
advantage of this approach over molecular MS-based peptide-
and protein-centric workflows is that only one generic sulfur-
containing standard is sufficient to quantify all of the proteins of
a proteome provided the components are sufficiently separated
and their amino acid sequences are known. The trend toward
hybrid mass analyzer configurations has dominated recent
advances in instrumentation. Current hybrid molecular mass
spectrometry systems combine the complementary perform-
ances offered by in-space beam-type and in-time ion-trapping
spectrometers into one instrument.
85
However, there are no
hybrid elemental and molecular mass spectrometry config-
urations on the market. In this work, we report a novel hybrid
instrumental setup to quantify the venom proteomes of the two
species of the genus Walterinnesia. Along with previous work on
the absolute quantification of other snake venom proteomes, it
highlights the feasibility of incorporating ICP-MS into hybrid
workflows that combine the unique performance of molecular
and elemental mass spectrometry, e.g., the unparalleled
molecular resolution of top-down MS and the absolute
quantification of ICP-MS. Our present work also validates our
long-standing strategy for the relative quantification of snake
venom proteomes (snake venomics), which primarily estimates
the relative abundances of the chromatographically separated
fractions as % by weight of the venom toxins/toxin families.
32
Analytical technological advances have continuously en-
hanced research on venoms. We would like to think that the
analytical advances discussed here toward absolute quantifica-
tion of snake venom proteomes of moderate complexity may
serve as a proof-of-concept for a broader and more routine
application of hybrid elemental/molecular MS setups in other
areas of the proteomics field Figure 5.
■ASSOCIATED CONTENT
*
sıSupporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jproteome.1c00608.
Transcriptomics database (Table S1); MS/MS identi-
fication of peptides/proteins in the RP-HPLC fractions of
the venom of adult W. aegyptia (Sinai Peninsula, Egypt)
(Table S2); MS/MS identification of peptides/proteins in
the RP-HPLC fractions of the venom of adult W. aegyptia
(Riyadh, Saudi Arabia) (Table S3); MS/MS identifica-
tion of peptides/proteins in the RP-HPLC fractions of the
venom of adult W. morgani (Corten village, Turkey)
(Table S4); top-down MS identifications of proteins in
the proteomes of W. aegyptia and W. morgani venoms
(Table S5); quantification by QQQ ICP-MS of the
venom proteome of W. aegyptia (Sinai Peninsula, Egypt)
(Table S6); quantification by QQQ ICP-MS of the
venom proteome of W. aegyptia (Riyadh, Saudi Arabia)
(Table S7); quantification by QQQ ICP-MS of the
venom proteome of W. morgani (Corten village, Turkey)
(Table S8) (XLSX)
■AUTHOR INFORMATION
Corresponding Author
Juan J. Calvete −Laboratorio de Venómica Evolutiva y
Traslational, Instituto de Biomedicina de Valencia, Consejo
Superior de Investigaciones Científicas (CSIC), 46010
Valencia, Spain; orcid.org/0000-0001-5026-3122;
Email: [email protected]
Authors
Davinia Pla −Laboratorio de Venómica Evolutiva y
Traslational, Instituto de Biomedicina de Valencia, Consejo
Superior de Investigaciones Científicas (CSIC), 46010
Valencia, Spain
Johannes Els −Environment and Protected Areas Authority,
82828 Sharjah, United Arab Emirates
Salvador Carranza −Institute of Evolutionary Biology, CSIC-
Universitat Pompeu Fabra, 08003 Barcelona, Spain
Maik Damm −Department of Chemistry, Technische
Universität Berlin, 10623 Berlin, Germany
Journal of Proteome Research pubs.acs.org/jpr Article
https://doi.org/10.1021/acs.jproteome.1c00608
J. Proteome Res. 2021, 20, 5064−5078
5074
Benjamin-Florian Hempel −Department of Chemistry,
Technische Universität Berlin, 10623 Berlin, Germany; BIH
Center for Regenerative Therapies BCRT, Charité-
Universitätsmedizin Berlin, 13353 Berlin, Germany
Elisa B. O. John −Center of Biotechnology, Universidade
Federal do Rio Grande do Sul, CEP 91501-970 Porto Alegre,
RS, Brazil
Daniel Petras −CMFI Cluster of Excellence, Interfaculty
Institute of Microbiology and Medicine, University of
Tubingen, 72076 Tubingen, Germany
Paul Heiss −Department of Chemistry, Technische Universität
Berlin, 10623 Berlin, Germany
Ayse Nalbantsoy −Department of Bioengineering, Faculty of
Engineering, Ege University, 35100 Bornova, Izmir, Turkey
Bayram Göcmen −Zoology Section, Department of Biology,
Faculty of Science, Ege University, 35100 Bornova, Izmir,
Turkey
Roderich D. Sussmuth −Department of Chemistry, Technische
Universität Berlin, 10623 Berlin, Germany
Francisco Calderón-Celis −Department of Physical and
Analytical Chemistry, University of Oviedo, 33006 Oviedo,
Spain
Alicia Jiménez Nosti −Department of Physical and Analytical
Chemistry, University of Oviedo, 33006 Oviedo, Spain
Jorge Ruiz Encinar −Department of Physical and Analytical
Chemistry, University of Oviedo, 33006 Oviedo, Spain;
orcid.org/0000-0001-6245-5770
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jproteome.1c00608
Notes
The authors declare no competing financial interest.
The bottom-up mass spectrometry proteomics data have been
deposited to the ProteomeXchange Consortium via the
PRIDE
86
partner repository with the dataset identifiers
PXD027495 (Venomics of the desert black cobra W. aegyptia
from Sinai Peninsula, Egypt), PXD027498 (Venomics of the
desert black cobra W. aegyptia from Riyadh, Saudi Arabia), and
PXD027497 (Venomics of the desert black cobra W. morgani
from Corten village, Turkey). Top-down LC−MS/MS.raw and
centroid.mzXML data are publicly available in the MassIVE
repository under the accession number MSV000086709. LC−
MS/MS data can be directly visualized through the GNPS LC−
MS dashboard (https://www.biorxiv.org/content/10.1101/
2021.04.05.438475v2)andtheLC−MS dataset explorer:
https://gnps-dataset-explorer.herokuapp.com/ with the Mas-
sive identifier MSV000086709 and under the following link:
https://gnps-lcms.ucsd.edu/?xicmz=980.8817%3B792.
24755859375&xic_formula=&xic_peptide=&xic_tolerance=0.
5&xic_ppm_tolerance=10&xic_tolerance_unit=Da&xic_rt_
window=&xic_norm=False&xic_file_grouping=GROUP&xic_
integration_type=AUC&show_ms2_markers=True&ms2_
identifier=MS2%3A1729&show_lcms_2nd_map=
True&map_plot_zoom=%7B%7D&polarity_filtering=
None&polarity_filtering2=None&tic_option=TIC&overlay_
usi=None&overlay_mz=row+m%2Fz&overlay_rt=
row+retention+time&overlay_color=&overlay_size=
&overlay_hover=&overlay_filter_column=&overlay_filter_
value=&feature_finding_type=Off&feature_finding_ppm=
10&feature_finding_noise=10000&feature_finding_min_
peak_rt=0.05&feature_finding_max_peak_rt=1.5&feature_
finding_rt_tolerance=0.
3#{%22usi%22:%20%22mzspec:MSV000086709:peak/27_
Walterinnesia_egyptia_Liverpool_unkown_red_2.mzXML\
nmzspec:MSV000086709:peak/27_Walterinnesia_egyptia_
Liverpool_unkown_red_1.mzXML\
n%22,%20%22usi2%22:%20%22mzspec:MSV000086709:
peak/30_Walterinnesia_morgani_Ayse_Turkey_red_1.
mzXML\nmzspec:MSV000086709:peak/30_Walterinnesia_
morgani_Ayse_Turkey_red_2.mzXML%22}.
■ACKNOWLEDGMENTS
This paper is dedicated to the memory of Prof. Bayram Göcmen,
a leading Turkish zoologist and passionate herpetologist, who
succumbed to cancer on 22 March 2019 at the early age of 54.
The authors wish to thank His Highness Sheikh Dr. Sultan bin
Mohammed Al Qasimi, Supreme Council Member and Ruler of
Sharjah, Her Excellency Hana Saif al Suwaidi (Chairperson,
Environment and Protected Areas Authority, Sharjah), Paul
Vercammen and Kevin Budd (Breeding Centre for Endangered
Arabian Wildlife) for their continuous support. S.C. was
supported by PGC2018-098290-B-I00 (MCIU/AEI/FEDER,
UE), Madrid, Spain. Research performed at IBV-CSIC and
University of Oviedo was partially funded by grants BFU2017-
89103-P and PID2019-109698GB-I00, respectively, from the
Ministerio de Ciencia e Innovación, Madrid, Spain (J.J.C.). This
work was also financed with funds from the Technische
Universität Berlin by the Department of International Scientific
Cooperation. Support by Agilent Technologies is also gratefully
acknowledged.
■REFERENCES
(1) Jenner, R.; Undheim, E. VenomThe Secrets of Nature’s Deadliest
Weapon; The Natural History Museum: London, U.K., 2017. ISBN
9780565094034.
(2) Schendel, V.; Rash, L. D.; Jenner, R. A.; Undheim, E. The diversity
of venom: the importance of behavior and venom system morphology
in understanding its ecology and evolution. Toxins 2019,11, No. 666.
(3) Van Valen, L. A new evolutionary law. Evol. Theory 1973,1,1−30.
(4) Dawkins, R.; Krebs, J. R. Arms races between and within species.
Proc. R. Soc. London, Ser. B 1979,205, 489−511.
(5) Mukherjee, S.; Heithaus, M. R. Dangerous prey and daring
predators: a review. Biol. Rev. Cambridge Philos. Soc. 2013,88, 550−563.
(6) Calvete, J. J. Venomics: integrative venom proteomics and beyond.
Biochem. J. 2017,474, 611−634.
(7) Evans, E. R. J.; Northfield, T. D.; Daly, N. L.; Wilson, D. T. Venom
Costs and Optimization in Scorpions. Front. Ecol. Evol. 2019,7,
No. 196.
(8) Glaudas, X.; Glennon, K. L.; Martins, M.; Luiselli, L.; Fearn, S.;
Trembath, D. F.; Jelic, D.; Alexander, G. J. Foraging mode, relative prey
size and diet breadth: A phylogenetically explicit analysis of snake
feeding ecology. J. Anim. Ecol. 2019,88, 757−767.
(9) Ward-Smith, H.; Arbuckle, K.; Naude, A.; Wuster, W. Fangs for
the Memories? A Survey of Pain in Snakebite Patients Does Not
Support a Strong Role for Defense in the Evolution of Snake Venom
Composition. Toxins 2020,12, No. 201.
(10) Gulick, S. P. S.; Bralower, T. J.; Ormö, J.; Hall, B.; Grice, K.;
Schaefer, B.; Lyons, S.; Freeman, K. H.; Morgan, J. V.; Artemieva, N.;
Kaskes, P.; de Graaff, S. J.; Whalen, M. T.; Collins, G. S.; Tikoo, S. M.;
Verhagen, C.; Christeson, G. L.; Claeys, P.; Coolen, M. J. L.; Goderis,
S.; Goto, K.; Grieve, R. A. F.; McCall, N.; Osinski, G. R.; Rae, A. S. P.;
Riller, U.; Smit, J.; Vajda, V.; Wittmann, A. Expedition 364 Scientists,
The first day of the Cenozoic. Proc. Natl. Acad. Sci. U.S.A. 2019,116,
19342−19351.
(11) Chiarenza, A. A.; Farnsworth, A.; Mannion, P. D.; Lunt, D. J.;
Valdes, P. J.; Morgan, J. V.; Allison, P. A. Asteroid impact, not
volcanism, caused the end-Cretaceous dinosaur extinction. Proc. Natl.
Acad. Sci. U.S.A. 2020,117, 17084−17093.
Journal of Proteome Research pubs.acs.org/jpr Article
https://doi.org/10.1021/acs.jproteome.1c00608
J. Proteome Res. 2021, 20, 5064−5078
5075
(12) Fry, B. G.; Casewell, N. R.; Wuster, W.; Vidal, N.; Young, B.;
Jackson, T. N. W. The structural and functional diversification of the
Toxicofera reptile venom system. Toxicon 2012,60, 434−448.
(13) Hsiang, A. Y.; Field, D. J.; Webster, T. H.; Behlke, A. D.; Davis,
M. B.; Racicot, R. A.; Gauthier, J. A. The origin of snakes: revealing the
ecology, behavior, and evolutionary history of early snakes using
genomics, phenomics, and the fossil record. BMC Evol. Biol. 2015,15,
No. 87.
(14) Calvete, J. J. Snake venomics: from the inventory of toxins to
biology. Toxicon 2013,75,44−62.
(15) Junqueira-de-Azevedo, I. L.; Campos, P. F.; Ching, A. T.;
Mackessy, S. P. Colubrid Venom Composition: An Omics Perspective.
Toxins 2016,8, No. 230.
(16) Tasoulis, T.; Isbister, G. K. A review and database of snake venom
proteomes. Toxins 2017,9, No. E290.
(17) Gutiérrez, J. M.; Calvete, J. J.; Habib, A. G.; Harrison, R. A.;
Williams, D. J.; Warrell, D. A. Snakebite envenoming. Nat. Rev. Dis.
Primers 2017,3, No. 17063.
(18) Barua, A.; Mikheyev, A. S. Many Options, Few Solutions: Over
60 My Snakes Converged on a Few Optimal Venom Formulations. Mol.
Biol. Evol. 2019,36, 1964−1974.
(19) Harrison, R. A.; Hargreaves, A.; Wagstaff, S. C.; Faragher, B.;
Lalloo, D. G. Snake envenoming: a disease of poverty. PLoS Neglected
Trop. Dis. 2009,3, No. e569.
(20) Longbottom, J.; Shearer, F. M.; Devine, M.; Alcoba, G.;
Chappuis, F.; Weiss, D. J.; Ray, S. E.; Ray, N.; Warrell, D. A.; Ruiz de
Castaneda, R.; Williams, D. J.; Hay, S. I.; Pigott, D. M. Vulnerability to
snakebite envenoming: a global mapping of hotspots. Lancet 2018,392,
673−684.
(21) Gutiérrez, J. M. Snakebite envenoming from an Ecohealth
perspective. Toxicon: X 2020,7, No. 100043.
(22) Jackson, T. N. W.; Jouanne, H.; Vidal, N. Snake Venom in
Context: Neglected Clades and Concepts. Front. Ecol. Evol. 2019,7,
No. 332.
(23) Calvete, J. J.; Lomonte, B.; Saviola, A. J.; Bonilla, F.; Sasa, M.;
Williams, D. J.; Undheim, E. A. B.; Sunagar, K.; Jackson, T. N. W.
Mutual enlightenment: a toolbox of concepts and methods for
integrating evolutionary and clinical toxinology via snake venomics
and the contextual stance. Toxicon: X 2021,9−10, No. 100070.
(24) Lomonte, B.; Rey-Suárez, P.; Fernández, J.; Sasa, M.; Pla, D.;
Vargas, N.; Bénard-Valle, M.; Sanz, L.; Correa-Netto, C.; Nunez, V.;
Alape-Girón, A.; Alagón, A.; Gutiérrez, J. M.; Calvete, J. J. Venoms of
Micrurus coral snakes: Evolutionary trends in compositional patterns
emerging from proteomic analyses. Toxicon 2016,122,7−25.
(25) Ainsworth, S.; Petras, D.; Engmark, M.; Sussmuth, S. D.;
Whiteley, G.; Albulescu, L. O.; Kazandjian, T. D.; Wagstaff, S. C.;
Rowley, P.; Wuster, W.; Dorrestein, P. C.; Arias, A. S.; Gutiérrez, J. M.;
Harrison, R. A.; Casewell, N. R.; Calvete, J. J. The medical threat of
mamba envenoming in sub-Saharan Africa revealed by genus-wide
analysis of venom composition, toxicity and antivenomics profiling of
available antivenoms. J. Proteomics 2018,172, 173−189.
(26) Calvete, J. J. Snake venomics at the crossroads between ecological
and clinical toxinology. Biochemist 2019,41,28−33.
(27) Holding, M. L.; Strickland, J. L.; Rautsaw, R. M.; Hofmann, E. P.;
Mason, A. J.; Hogan, M. P.; Nystrom, G. S.; Ellsworth, S. A.; Colston, T.
J.; Borja, M.; Castaneda-Gaytán, G.; Grunwald, C. I.; Jones, J. M.;
Freitas-de-Sousa, L. A.; Viala, V. L.; Margres, M. J.; Hingst-Zaher, E.;
Junqueira-de-Azevedo, I. L. M.; Moura-da-Silva, A. M.; Grazziotin, F.
G.; Gibbs, H. L.; Rokyta, D. R.; Parkinson, C. L. Phylogenetically
diverse diets favor more complex venoms in North American pitvipers.
Proc. Natl. Acad. Sci. U.S.A. 2021,118, No. e2015579118.
(28) The Reptile Database, 2021. http://www.reptile-database.org.
(29) Damm, M.; Hempel, B. F.; Sussmuth, R. D. Old World Vipers-A
Review about Snake Venom Proteomics of Viperinae and Their
Variations. Toxins 2021,13, No. 427.
(30) Lomonte, B.; Calvete, J. J. Strategies in ‘snake venomics’aiming
at an integrative view of compositional, functional, and immunological
characteristics of venoms. J. Venomous Anim. Toxins Incl. Trop. Dis.
2017,23, No. 26.
(31) Calvete, J. J.; Petras, D.; Calderón-Celis, J.; Lomonte, B.; Ruiz
Encinar, J.; Sanz-Medel, A. Protein-species quantitative venomics:
looking through a crystal ball. J. Venomous Anim. Toxins Incl. Trop. Dis.
2017,23, No. 27.
(32) Calvete, J. J. Snake venomics-from low-resolution toxin-pattern
recognition to toxin-resolved venom proteomes with absolute
quantification. Expert Rev. Proteomics 2018,15, 555−568.
(33) Quantitative Proteomics: New Developments in Mass Spectrometry;
Eyers, C. E.; Gaskell, S., Eds.; RSC Publishing, 2014. ISBN 978-1-
84973-808-8.
(34) Urban, P. L. Quantitative mass spectrometry: an overview. Philos.
Trans. R. Soc., A 2016,374, No. 20150382.
(35) Picard, G.; Lebert, D.; Louwagie, M.; Adrait, A.; Huillet, C.;
Vandenesch, F.; Bruley, C.; Garin, J.; Jaquinod, M.; Brun, V. PSAQ
standards for accurate MS-based quantification of proteins: from the
concept to biomedical applications. J. Mass Spectrom. 2012,47, 1353−
1363.
(36) Calderón-Celis, F.; Ruiz Encinar, J.; Sanz-Medel, A. Stand-
ardization approaches in absolute quantitative proteomics with mass
spectrometry. Mass Spectrom. Rev. 2018,37, 715−737.
(37) Brun, V.; Masselon, C.; Garin, J.; Dupuis, A. Isotope dilution
strategies for absolute quantitative proteomics. J. Proteomics 2009,72,
740−749.
(38) Alonso, J.; González, P. Isotope Dilution Mass Spectrometry; RSC
Publishing, 2013. ISBN 978-1-84973-333-5.
(39) Villanueva, J.; Carrascal, M.; Abian, J. Isotope dilution mass
spectrometry for absolute quantification in proteomics: concepts and
strategies. J. Proteomics 2014,96, 184−199.
(40) Calderón-Celis, F.; Diez-Fernández, S.; Costa-Fernández, J. M.;
Ruiz Encinar, J.; Calvete, J.; Sanz-Medel, A. Elemental Mass
Spectrometry for Absolute Intact Protein Quantification without
Protein-Specific Standards: Application to Snake Venomics. Anal.
Chem. 2016,88, 9699−9706.
(41) Calderón-Celis, F.; Cid-Barrio, L.; Ruiz Encinar, J.; Sanz-Medel,
A.; Calvete, J. J. Absolute venomics: Absolute quantification of intact
venom proteins through elemental mass spectrometry. J. Proteomics
2017,164,33−42.
(42) Calderón-Celis, F.; Sanz-Medel, A.; Ruiz Encinar, J. Universal
absolute quantification of biomolecules using element mass spectrom-
etry and generic standards. Chem. Commun. 2018,54, 904−907.
(43) Calderón-Celis, F.; Sugiyama, N.; Yamanaka, M.; Sakai, T.; Diez-
Fernández, S.; Calvete, J. J.; Sanz Medel, A.; Ruiz Encinar, J. Enhanced
Universal Quantification of Biomolecules Using Element MS and
Generic Standards: Application to Intact Protein and Phosphoprotein
Determination. Anal. Chem. 2019,91, 1105−1112.
(44) Lataste, F. Description d’un nouveau genre et d’une nouvelle
espece d’ophidien proteroglyphe d’Egypte. Le Nat. 1887,9, 411−413.
(45) Mocquard, M. F. Diagnoses de quelques especes nouvelles de
Reptiles. Bull. Mus. Natl. Hist. Nat. 1905,11,76−79.
(46) Nilson, G.; Rastegar-Pouyani, N. Walterinnesia aegyptia Lataste,
1887 (Ophidia: Elapidae) and the status of Naja morgani Mocquard
1905. Russian J. Herpetol. 2007,14,7−14.
(47) Haas, G.; Werner, Y. L. Lizards and snakes from southwestern
Asia. Bull. Mus. Comp. Zool. 1969,139, 327−406.
(48) Joger, U. The Venomous Snakes of the Near and Middle East, No.
12; Reihe, A., Eds.; Beihefte zum Tubinger Atlas des Vorderen Orients,
1984.
(49) Gasperetti, J. Snakes of Arabia; Fauna of Saudi Arabia: Berne,
Riyad, 1988; Vol. 9, pp 169−450.
(50) Spawls, S.; Branch, B. The Dangerous Snakes of Africa;
Bloomsbury Publishing Plc: London, U.K., 2020. ISBN 978-1-4729-
6026-9.
(51) Al-Sadoon, M. K.; Fahim, A.; Salama, S. F.; Badr, G. The effects of
LD50 of Walterinnesia aegyptia crude venom on blood parameters of
male rats. African. Afr. J. Microbiol. Res. 2012,6, 653−659.
(52) Amr, Z. R.; Abu Baker, M. A.; Warrell, D. A. Terrestrial
venomous snakes and snakebites in the Arab countries of the Middle
East. Toxicon 2020,177,1−15.
Journal of Proteome Research pubs.acs.org/jpr Article
https://doi.org/10.1021/acs.jproteome.1c00608
J. Proteome Res. 2021, 20, 5064−5078
5076
(53) Lauer, C.; Zickgraf, T. L.; Weisse, M. E. Case report of probable
desert black snake envenomation in 22-year-old male causing profound
weakness and respiratory distress. Wilderness Environ. Med. 2011,22,
246−249.
(54) Tamar, K.; Chirio, L.; Shobrak, M.; Busais, S.; Carranza, S. Using
multilocus approach to uncover cryptic diversity within Pseudotrapelus
lizards from Saudi Arabia. Saudi J. Biol. Sci. 2019,26, 1442−1449.
(55) Tamar, K.; Els, J.; Kornilios, P.; Soorae, P.; Tarroso, P.; Thanou,
E.; Pereira, J.; Shah, J. N.; Elhassan, E. E. M.; Aguhob, J. C.; Badaam, S.
F.; Eltayeb, M. M.; Pusey, R.; Papenfuss, T. J.; Macey, J. R.; Carranza, S.
The demise of a wonder: Evolutionary history and conservation
assessments of the Wonder Gecko Teratoscincus keyserlingii (Gekkota,
Sphaerodactylidae) in Arabia. PLoS One 2021,16, No. e0244150.
(56) Göcmen, B.; Franzen, M.; Yildiz, M. Z.; Akman, B.; Yalcınkaya,
D. New locality records of eremial snake species in southeastern Turkey
(Ophidia: Colubridae, Elapidae, Typhlopidae, Leptotyphlopidae).
Salamandra 2009,45, 110−114.
(57) OECD TN. Acute Oral Toxicity Up-and-Down-Procedure
(UDP). In OECD Guidelines for the Testing of Chemicals; OECD, 2008;
Vol. 425,pp1−27.
(58) Petras, D.; Hempel, B. F.; Göcmen, B.; Karis, M.; Whiteley, G.;
Wagstaff, S. C.; Heiss, P.; Casewell, N. R.; Nalbantsoy, A.; Sussmuth, R.
D. Intact protein mass spectrometry reveals intraspecies variations in
venom composition of a local population of Vipera kaznakovi in
Northeastern Turkey. J. Proteomics 2019,199,31−50.
(59) Calvete, J. J. Proteomic tools against the neglected pathology of
snake bite envenoming. Expert Rev. Proteomics 2011,8, 739−758.
(60) Eichberg, S.; Sanz, L.; Calvete, J. J.; Pla, D. Constructing
comprehensive venom proteome reference maps for integrative
venomics. Expert Rev. Proteomics 2015,12, 557−573.
(61) Pla, D.; Sanz, L.; Quesada-Bernat, S.; Villalta, M.; Baal, J.;
Chowdhury, M. A. W.; León, G.; Gutiérrez, J. M.; Kuch, U.; Calvete, J. J.
Phylovenomics of Daboia russelii across the Indian subcontinent.
Bioactivities and comparative in vivo neutralization and in vitro third-
generation antivenomics of antivenoms against venoms from India,
Bangladesh and Sri Lanka. J. Proteomics 2019,207, No. 103443.
(62) Sánchez, A.; Segura, A.; Pla, D.; Munuera, J.; Villalta, M.;
Quesada-Bernat, S.; Chavarría, D.; Herrera, M.; Gutiérrez, J. M.; León,
G.; Calvete, J. J.; Vargas, M. Comparative venomics and preclinical
efficacy evaluation of a monospecific Hemachatus antivenom towards
sub-Saharan Africa cobra venoms. J. Proteomics 2021,240, No. 104196.
(63) Calvete, J. J. Next-generation snake venomics: protein-locus
resolution through venom proteome decomplexation. Expert Rev.
Proteomics 2014,11, 315−329.
(64) Petras, D.; Heiss, P.; Harrison, R. A.; Sussmuth, R. D.; Calvete, J.
J. Top-down venomics of the East African green mamba, Dendroaspis
angusticeps, and the black mamba, Dendroaspis polylepis, highlight the
complexity of their toxin arsenals. J. Proteomics 2016,146, 148−164.
(65) Hempel, B. F.; Damm, M.; Mrinalini; Göcmen, B.; Karıs, M.;
Nalbantsoy, A.; Kini, R. M.; Sussmuth, R. D. Extended Snake Venomics
by Top-Down In-Source Decay: Investigating the Newly Discovered
Anatolian Meadow Viper Subspecies, Vipera anatolica senliki. J.
Proteome Res. 2020,19, 1731−1749.
(66) Pla, D.; Petras, D.; Saviola, A. J.; Modahl, C. M.; Sanz, L.; Pérez,
A.; Juárez, E.; Frietze, S.; Dorrestein, P. C.; Mackessy, S. P.; Calvete, J. J.
Transcriptomics-guided bottom-up and top-down venomics of neonate
and adult specimens of the arboreal rear-fanged Brown Treesnake,
Boiga irregularis, from Guam. J. Proteomics 2018,174,71−84.
(67) Chambers, M. C.; Maclean, B.; Burke, R.; Amodei, D.;
Ruderman, D. L.; Neumann, S.; Gatto, L.; Fischer, B.; Pratt, B.;
Egertson, J.; Hoff, K.; Kessner, D.; Tasman, N.; Shulman, N.; Frewen,
B.; Baker, T. A.; Brusniak, M. Y.; Paulse, C.; Creasy, D.; Flashner, L.;
Kani, K.; Moulding, C.; Seymour, S. L.; Nuwaysir, L. M.; Lefebvre, B.;
Kuhlmann, F.; Roark, J.; Rainer, P.; Detlev, S.; Hemenway, T.; Huhmer,
A.; Langridge, J.; Connolly, B.; Chadick, T.; Holly, K.; Eckels, J.;
Deutsch, E. W.; Moritz, R. L.; Katz, J. E.; Agus, D. B.; MacCoss, M.;
Tabb, D. L.; Mallick, P. A cross-platform toolkit for mass spectrometry
and proteomics. Nat. Biotechnol. 2012,30, 918−920.
(68) Kou, Q.; Xun, L.; Liu, X. TopPIC: a software tool for top-down
mass spectrometry-based proteoform identification and character-
ization. Bioinformatics 2016,32, 3495−3497.
(69) Diez Fernández, S.; Sugishama, N.; Ruiz Encinar, J.; Sanz-Medel,
A. Triple quad ICPMS (ICPQQQ) as a new tool for absolute
quantitative proteomics and phosphoproteomics. Anal. Chem. 2012,84,
5851−5857.
(70) Wind, M.; Wegener, A.; Eisenmenger, A.; Kelner, R.; Lehmann,
W. D. Sulfur as the Key Element for Quantitative Protein Analysis by
Capillary Liquid Chromatography Coupled to Element Mass
Spectrometry. Angew. Chem., Int. Ed. 2003,42, 3425−3427.
(71) Cid-Barrio, L.; Calderón-Celis, F.; Costa-Fernández, J. M.; Ruiz
Encinar, J. Assessment of the Potential and Limitations of Elemental
Mass Spectrometry in Life Sciences for Absolute Quantification of
Biomolecules Using Generic Standards. Anal. Chem. 2020,92, 13500−
13508.
(72) Calderón-Celis, F.; Ruiz Encinar, J. A reflection on the role of
ICP-MS in proteomics: Update and future perspective. J. Proteomics
2019,198,11−17.
(73) Kazandjian, T. D.; Petras, D.; Robinson, S. D.; van Thiel, J.;
Greene, H. W.; Arbuckle, K.; Barlow, A.; Carter, D. A.; Wouters, R. M.;
Whiteley, G.; Wagstaff, S. C.; Arias, A. S.; Albulescu, L. O.; Plettenberg
Laing, A.; Hall, C.; Heap, A.; Penrhyn-Lowe, S.; McCabe, C. V.;
Ainsworth, S.; da Silva, R. R.; Dorrestein, P. C.; Richardson, M. K.;
Gutiérrez, J. M.; Calvete, J. J.; Harrison, R. A.; Vetter, I.; Undheim, E. A.
B.; Wuster, W.; Casewell, N. R. Convergent evolution of pain-inducing
defensive venom components in spitting cobras. Science 2021,371,
386−390.
(74) Melani, R. D.; Skinner, O. S.; Fornelli, L.; Domont, G. B.;
Compton, P. D.; Kelleher, N. L. Mapping Proteoforms and Protein
Complexes From King Cobra Venom Using Both Denaturing and
Native Top-down Proteomics. Mol. Cell. Proteomics 2016,15, 2423−
2434.
(75) Quantitative Methods in Proteomics. In Methods in Molecular
Biology, 2228, 2nd ed.; Markus, K.; Eisenacher, M.; Sitek, B., Eds.;
Humana Press: New York, 2021. ISBN 978-1-0716-1023-7.
(76) Jarnuczak, A. F.; Lee, D. C.; Lawless, C.; Holman, S. W.; Eyers, C.
E.; Hubbard, S. J. Analysis of Intrinsic Peptide Detectability via
Integrated Label-Free and SRM-Based Absolute Quantitative Proteo-
mics. J. Proteome Res. 2016,15, 2945−2959.
(77) Li, Y. F.; Arnold, R. J.; Tang, H.; Radivojac, P. The importance of
peptide detectability for protein identification, quantification, and
experiment design in MS/MS proteomics. J. Proteome Res. 2010,9,
6288−6297.
(78) Mohammed, Y.; Pan, J.; Zhang, S.; Han, J.; Borchers, C. ExSTA:
external standard addition method for accurate high-throughput
quantitation in targeted proteomics experiments. Proteomics: Clin.
Appl. 2008,12, No. 1600180.
(79) Sanz-Medel, A.; Montes-Bayón, M.; Fernández Sánchez, M. L.
Trace element speciation by ICP-MS in large biomolecules and its
potential for proteomics. Anal. Bioanal. Chem. 2003,377, 236−247.
(80) Sanz-Medel, A. “Heteroatom-tagged”quantification of proteins
via ICP-MS. Anal. Bioanal. Chem. 2016,408, 5393−5395.
(81) Bettmer, J.; Montes Bayón, M.; Ruiz Encinar, J.; Fernández
Sánchez, M. L.; Fernández de la Campa, M. R.; Sanz Medel, A. The
emerging role of ICP-MS in proteomic analysis. J. Proteomics 2009,72,
989−1005.
(82) Kuipers, B. J.; Gruppen, H. Prediction of molar extinction
coefficients of proteins and peptides using UV absorption of the
constituent amino acids at 214 nm to enable quantitative reverse phase
high-performance liquid chromatography-mass spectrometry analysis.
J. Agric. Food Chem. 2007,55, 5445−5451.
(83) Houk, R. S.; Tassel, V. A.; Flesch, G. D.; Svec, H. J.; Gray, A. L.;
Taylor, C. E. Inductively coupled argon plasma as an ion source for
mass spectrometric determination of trace elements. Anal. Chem. 1980,
53, 2283−2289.
(84) Becker, J. S. Inorganic Mass Spectrometry. In Principles and
Applications; John Wiley & Sons Ltd.: Chichester, U.K., 2007; pp 118−
176. ISBN 978-0-470-01200-0.
Journal of Proteome Research pubs.acs.org/jpr Article
https://doi.org/10.1021/acs.jproteome.1c00608
J. Proteome Res. 2021, 20, 5064−5078
5077
(85) Calvete, J. J. The Expanding Universe of Mass Analyzer
Configurations for Biological Analysis. In Plant Proteomics: Methods and
Protocols, Methods in Molecular Biology; Springer Science + Business
Media, LLC, 2014; Vol. 1072,pp61−81.
(86) Perez-Riverol, Y.; Csordas, A.; Bai, J.; Bernal-Llinares, M.;
Hewapathirana, S.; Kundu, D. J.; Inuganti, A.; Griss, J.; Mayer, G.;
Eisenacher, M.; Pérez, E.; Uszkoreit, J.; Pfeuffer, J.; Sachsenberg, T.;
Yilmaz, S.; Tiwary, S.; Cox, J.; Audain, E.; Walzer, M.; Jarnuczak, A. F.;
Ternent, T.; Brazma, A.; Vizcaíno, J. A. The PRIDE database and
related tools and resources in 2019: improving support for
quantification data. Nucleic Acids Res. 2019,47, D442−D450.
Journal of Proteome Research pubs.acs.org/jpr Article
https://doi.org/10.1021/acs.jproteome.1c00608
J. Proteome Res. 2021, 20, 5064−5078
5078
