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Anal Chem. 2010 Feb 15;82(4):1234-44. doi: 10.1021/ac9021083.

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

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1
Department of Chemistry, 600 South Mathews Avenue, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.

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.

PMID:
20073486
PMCID:
PMC2823583
DOI:
10.1021/ac9021083
[Indexed for MEDLINE]
Free PMC Article
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