Bruker TimsTof Ultra acquisition and spectrum matching
Abstract
This is a summary of the settings used for the MS acquisition using Bruker Tims Tof ULTRA in the DIA mode and subsequent spectrum matching with the DIA-NN software. All the settings can be found in TTofUltra-DIA-DiaNN-autonomics.md . If any alterations to these settings were made, it is explicitly specified in the manuscript. MS acquisition method can be extracted from dia_norm_0.96s.proteoscape.m.zip .
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TimsTof Ultra, nanoElute, Dia-NN default settings Witold Szymanski, Johannes Graumann 2025-11-07 Table of contents 1 Instrumentation 2 2 LC methods 2 2.1 twoCol methods (with trap-column) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 2.1.1 twoCol - 30 min gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 2.1.2 twoCol - 45 min gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 2.1.3 twoCol - 60 min gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 2.1.4 twoCol - 90 min gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 2.2 oneColmethods ............................................. 7 2.2.1 oneCol - 30 min gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 2.2.2 oneCol - 45 min gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 2.2.3 oneCol - 60 min gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 2.2.4 oneCol - 90 min gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 3 MS acquisition methods 11 3.1 DIA-PASEF............................................... 11 3.1.1 dia-norm_0.96s ......................................... 11 3.1.2 dia-sens_0.96s.......................................... 13 4 Peptide spectrum matching and label-free quantitation 15 5 References: 15 1
1 Instrumentation • Mass Spectrometer: Bruker Daltonics timsTOF Ultra • Liquid Chromatography: nanoElute2 • trap column: Thermo Trap Cartridge 5mm, µ-Precolum TM Cartridge / PepMap TM C18, Thermo Scientific • analytical column: PepSep Ultra, C18, 1.5�μm, 75�μm�×�25�cm, Bruker Daltonics • emitter: 20 μm • source: CaptiveSpray Ultra 2 LC methods • default settings: –solvent A: 0.1% formic acid –solvent B: 99.9% acetonitrile and 0.1% formic acid –flow rate: 300 nL/min –50 ng of peptides per injection 2
2.1 twoCol methods (with trap-column) 2.1.1 twoCol - 30 min gradient Figure 1: twoCol - 30 min gradient 3
2.1.2 twoCol - 45 min gradient Figure 2: twoCol - 45 min gradient 4
2.1.3 twoCol - 60 min gradient Figure 3: twoCol - 60 min gradient 5
2.1.4 twoCol - 90 min gradient Figure 4: twoCol - 90 min gradient 6
2.2 oneCol methods 2.2.1 oneCol - 30 min gradient Figure 5: oneCol - 30 min gradient 7
2.2.2 oneCol - 45 min gradient Figure 6: oneCol - 45 min gradient 8
2.2.3 oneCol - 60 min gradient Figure 7: oneCol - 60 min gradient 9