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DMSO in NanoLC /MS. A case study on TMT-labeled human cell lysate digest. Acknowledgements. Experiments carried out by Henrik Johansson Rui Branca Karolinska Institutet Stockholm Data processing and report Michaela Scigelova Thermo Scientific. Introduction.
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DMSO in NanoLC/MS A case study on TMT-labeled human cell lysate digest
Acknowledgements • Experiments carried out by • Henrik Johansson • Rui Branca • KarolinskaInstitutet Stockholm • Data processing and report • Michaela Scigelova • Thermo Scientific
Introduction • Recent publication from B. Kuster‘s group reported an improved performance of nanoLC-MS analysis of complex tryptic peptide digests upon addition of 5% DMSO into the mobile phase • The effects observed include better spray stability, increased signal response of the MS, overall shift of charge state distribution for individual peptides towards lower values resulting in the predominance of 2+ species • Significant increase in the number of identifications • No negative effects have been noted
Goal • Evaluate the effect of using 5% DMSO in mobile phase in nanoLC/MS of complex peptide mixture on the following figures of merit • Signal intensity • Mass accuracy • Precursor charge state distribution • Methione oxidation state • Peptide and protein ID
Experiment Outline • Sample • Human cell line • Digested with trypsin, alkylated with iodoacetamide • Labeled with TMT 10plex • NanoLC separation • Agilent 1200 series • 4 h gradient • 400 nl/min flow rate • Q Exactive • Thermo Scientific • Top5 data dependent acquisition with dynamic exclusion • Proteome Discoverer 1.4 sw
Experimental Details: LC Method • Mobile Phase A: Water containing 0.1% formic acid - with/without 5% (v/v) DMSO • Mobile Phase B: 80/20 acetonitrile/water containing 0.1% formic acid – with/without 5% (v/v) DMSO • Loading buffer: 98/2 water/acetonitrile containing 0.05% TFA, flow rate 8 µl/min • Trap: 100µm ID x 2cm, 5µm • Column: 75µm ID x 15cm, 5µm C18 • Nanosource: Flex (Thermo Scientific)
Experimental Details: Q Exactive Method • TOP5 Runtime 0 to 255 min Polarity positive • Full MS Resolution 70,000 AGC target 1e6 Maximum IT 100 ms Scan range 300 to 1700 m/z Spectrum data type Profile • dd-MS² Resolution 35,000 AGC target 1e5 Maximum IT 500 ms Isolation window 2.0 m/z Fixed first mass 100.0 m/z NCE 30.0 Underfill ratio 1.0 % Peptide match preferred Exclude isotopes on Dynamic exclusion 60.0 s
Base Peak MS +DMSO +DMSO Judging from NL values, there does not seem to be any significant increase in MS signal
Precursor Mass Deviation • Two replicate injections (no DMSO) • Average mass deviation -0.12 ppm • Median mass deviation -0.13 ppm
Precursor Mass Deviation • Two replicate injections (added DMSO) • Average mass deviation -1.06 ppm • Median mass deviation -1.30 ppm Mass deviation slightly increased for DMSO runs Not significant / Inconclusive
Charge State Distribution • Distribution of charge states in duplicate runs with/without DMSO (5% v/v) 69% increase in 2+ charge state species
Peptide detected as 2+ only in ‘addedDMSO’ file • Identification of peptide aAATPESQEPQAk in noDMSO (A2) and addedDMSO (B2) sample noDMSO; 3+ addedDMSO; 2+
Peptide detected as 2+ only in ‘addedDMSO’ file • XIC for 2+ and 3+ aAATPESQEPQAk in noDMSO and addedDMSO sample noDMSO; 2+ noDMSO; 3+ addedDMSO; 2+ addedDMSO; 3+
Oxidative Effect of DMSO - MetOx • Proportion of oxidized Met-containing peptides (FDR 1%) Minor increase in frequency of Met oxidation
Peptide/Protein ID • Result of two replicate injections 3.7% increase in identified PSMs 17.2% increase in protein ID
Peptide ID • Comparison for 2 replicate injections (noDMSO) Overlap 56.7%
Peptide ID • Comparison for 2 replicate injections (addedDMSO) Overlap 58.3%
Peptide ID • Comparison for 1 injection each Overlap 40.7%
Summary and Conclusion • There is a considerably less overlap between peptides detected in noDMSOvsaddedDMSO samples when compared to an overlap in replicate injections of the same sample • 69% increase in the population of 2+ charge state peptides • 17.2% increase in protein ID • No significant increase in peptides with oxidized Met • No significant change in MS signal • No significant impact on mass accuracy A positive effect of DMSO on protein ID confirmed