1 / 21

DMSO in NanoLC /MS

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.

mahina
Download Presentation

DMSO in NanoLC /MS

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. DMSO in NanoLC/MS A case study on TMT-labeled human cell lysate digest

  2. Acknowledgements • Experiments carried out by • Henrik Johansson • Rui Branca • KarolinskaInstitutet Stockholm • Data processing and report • Michaela Scigelova • Thermo Scientific

  3. 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

  4. 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

  5. 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

  6. 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)

  7. 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

  8. Experimental Details: Data Processing

  9. Results

  10. Base Peak MS +DMSO +DMSO Judging from NL values, there does not seem to be any significant increase in MS signal

  11. Precursor Mass Deviation • Two replicate injections (no DMSO) • Average mass deviation -0.12 ppm • Median mass deviation -0.13 ppm

  12. 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

  13. Charge State Distribution • Distribution of charge states in duplicate runs with/without DMSO (5% v/v) 69% increase in 2+ charge state species

  14. Peptide detected as 2+ only in ‘addedDMSO’ file • Identification of peptide aAATPESQEPQAk in noDMSO (A2) and addedDMSO (B2) sample noDMSO; 3+ addedDMSO; 2+

  15. 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+

  16. Oxidative Effect of DMSO - MetOx • Proportion of oxidized Met-containing peptides (FDR 1%) Minor increase in frequency of Met oxidation

  17. Peptide/Protein ID • Result of two replicate injections 3.7% increase in identified PSMs 17.2% increase in protein ID

  18. Peptide ID • Comparison for 2 replicate injections (noDMSO) Overlap 56.7%

  19. Peptide ID • Comparison for 2 replicate injections (addedDMSO) Overlap 58.3%

  20. Peptide ID • Comparison for 1 injection each Overlap 40.7%

  21. 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

More Related