Size-sorting combined with improved nanocapillary liquid chromatography-mass spectrometry for identification of intact proteins up to 80 kda

Adaikkalam Vellaichamy, John C. Tran, Adam D. Catherman, Ji Eun Lee, John F. Kellie, Steve M. Steve, Leonid Zamdborg, Paul M. Thomas, Dorothy R. Ahlf, Kenneth R. Durbin, Gary A. Valaskovic, Neil L. Kelleher

Research output: Contribution to journalArticlepeer-review

77 Scopus citations

Abstract

Despite the availability of ultra-high-resolution mass spectrometers, methods for separation and detection of intact proteins for proteome-scale analyses are still in a developmental phase. Here we report robust protocols for online LC-MS to drive high-throughput top-down proteomics in a fashion similar to that of bottom-up proteomics. Comparative work on protein standards showed that a polymeric stationary phase led to superior sensitivity over a silica-based medium in reversed-phase nanocapillary LC, with detection of proteins > 50 kDa routinely accomplished in the linear ion trap of a hybrid Fourier transform mass spectrometer. Protein identification was enabled by nozzle-skimmer dissociation and detection of fragment ions with < 10 ppm mass accuracy for highly specific database searching using tailored software. This overall approach led to identification of proteins up to 80 kDa, with 10-60 proteins identified in single LC-MS runs of samples from yeast and human cell lines prefractionated by their molecular mass using a gel-based sieving system.

Original languageEnglish (US)
Pages (from-to)1234-1244
Number of pages11
JournalAnalytical Chemistry
Volume82
Issue number4
DOIs
StatePublished - Feb 15 2010

ASJC Scopus subject areas

  • Analytical Chemistry

Fingerprint Dive into the research topics of 'Size-sorting combined with improved nanocapillary liquid chromatography-mass spectrometry for identification of intact proteins up to 80 kda'. Together they form a unique fingerprint.

Cite this