Enhancing discovery and quantitative proteomics by use of online multiphase chip LC fractionation — ASN Events
  1. Schedule
  2. Lightning Talks Two
  3. Enhancing discovery and quantitative proteomics by use of online multiphase chip LC fractionation

Enhancing discovery and quantitative proteomics by use of online multiphase chip LC fractionation (#203)

Christoph Krisp 1 , Robert Parker 2 , Dana Pascovici 3 , Hao Yang 3 , Remco van Soest 3 , Mark P Molloy 1
  1. Australian Proteome Analysis Facility, Department of Chemistry & Biomolecular Sciences, Macquarie University, Sydney, New South Wales, Australia
  2. Department of Respiratory Medicine, Imperial College of Science, Technology and Medicine, London, United Kingdom
  3. Eksigent part of AB SCIEX, Redwood City, California, USA

Online strong cation exchange (SCX) coupled with reversed phase (RP) mass spectrometry (MS) also known as MudPIT (multi-dimensional protein identification technology) is widely used in discovery-based proteomics. We combined MudPIT with selected reaction monitoring (SRM) MS to avoid off-line sample handling and showed a significant boost in signal response (90% increase).1

Encouraged by these findings, we developed chip-based SCX coupled RP columns to achieve higher column-to-column reproducibility and greater robustness of automated LC-MS/MS workflows. Technical proofs were established using cell lysates and plasma (non-immunodepleted) digests (5 µg). Five-step salt-based fractionation followed by 1 h acetonitrile gradients revealed significant peptide identification increases compared with 1 h RP-LC-MS/MS (5 µg, ~80% increase) and more accumulated unique peptides compared with 5x 1 h RP-LC-MS/MS runs (25 µg, ~10% increase). Multiphase separated samples demonstrated remarkable elution consistency (less than 6% in more than 1 fraction in cell lysates, and less than 13% in plasma).

Highly reproducible separation enabled multiphase chip integration into high resolution multiple reaction monitoring (MRM-HR) applications. Fraction-specific and retention time scheduled peptide lists (503 peptides from 173 plasma proteins) were generated. All targeted peptides were quantifiable in multiphase MRM-HR while 16% (85 peptides) were undetectable in conventional MRM-HR and demonstrated 70% mean peak area increases. Interestingly, predominantly early eluting peptides demonstrated greatest increases (>10-fold).

Multiphase fractionation was also beneficial for spectral library generation in data independent quantitation (SWATH-MS). Five-step fractionation of thyroid cancer cell lysates led to spectral library generation for 3000 proteins. Analysing SWATH-MS acquisitions of same lysates resulted in reliable quantitation of 85% of all proteins in the library.

Using multiphase fractionation with SWATH-MS to analyse 10 melanoma cells with different MAPK pathway mutations demonstrated (principle component analysis) explicitly segregation of MEK inhibitor sensitive cells from MEK insensitive cells with distinct marker proteins for these phenotypes (confirmed by SRM-MS).

  1. (1) Krisp et al., Anal. Chem., 2012, 84(3), 1592–1600.