Size-sorting combined with improved nanocapillary liquid chromatography-mass spectrometry for identification of intact proteins up to 80 kDa.
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.
Vellaichamy
Adaikkalam
A
Department of Chemistry, 600 South Mathews Avenue, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.
Tran
John C
JC
Catherman
Adam D
AD
Lee
Ji Eun
JE
Kellie
John F
JF
Sweet
Steve M M
SM
Zamdborg
Leonid
L
Thomas
Paul M
PM
Ahlf
Dorothy R
DR
Durbin
Kenneth R
KR
Valaskovic
Gary A
GA
Kelleher
Neil L
NL
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D036103:Nanotechnology; D011108:Polymers / Q000737:chemistry; D016062:Porosity; D011506:Proteins / Q000032:analysis* / Q000737:chemistry*; D020543:Proteome / Q000032:analysis / Q000737:chemistry; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis / Q000737:chemistry; D013997:Time Factors
D011108:Polymers; D011506:Proteins; D020543:Proteome; D029701:Saccharomyces cerevisiae Proteins