Evaluation of multidimensional chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) for large-scale protein analysis: the yeast proteome.
Highly complex protein mixtures can be directly analyzed after proteolysis by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). In this paper, we have utilized the combination of strong cation exchange (SCX) and reversed-phase (RP) chromatography to achieve two-dimensional separation prior to MS/MS. One milligram of whole yeast protein was proteolyzed and separated by SCX chromatography (2.1 mm i.d.) with fraction collection every minute during an 80-min elution. Eighty fractions were reduced in volume and then re-injected via an autosampler in an automated fashion using a vented-column (100 microm i.d.) approach for RP-LC-MS/MS analysis. More than 162,000 MS/MS spectra were collected with 26,815 matched to yeast peptides (7,537 unique peptides). A total of 1,504 yeast proteins were unambiguously identified in this single analysis. We present a comparison of this experiment with a previously published yeast proteome analysis by Yates and colleagues (Washburn, M. P.; Wolters, D.; Yates, J. R., III. Nat. Biotechnol. 2001, 19, 242-7). In addition, we report an in-depth analysis of the false-positive rates associated with peptide identification using the Sequest algorithm and a reversed yeast protein database. New criteria are proposed to decrease false-positives to less than 1% and to greatly reduce the need for manual interpretation while permitting more proteins to be identified.
Peng
Junmin
J
Department of Cell Biology, and Taplin Biological Mass Spectrometry Facility, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA.
Elias
Joshua E
JE
Thoreen
Carson C
CC
Licklider
Larry J
LJ
Gygi
Steven P
SP
D002412:Cations; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange / Q000379:methods; D002853:Chromatography, Liquid / Q000379:methods*; D019992:Databases as Topic; D005189:False Positive Reactions; D005656:Fungal Proteins / Q000032:analysis / Q000737:chemistry*; D007477:Ions; D013058:Mass Spectrometry / Q000379:methods*; D036103:Nanotechnology; D010455:Peptides / Q000737:chemistry; D020543:Proteome; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000378:metabolism; D013997:Time Factors
D002412:Cations; D005656:Fungal Proteins; D007477:Ions; D010455:Peptides; D020543:Proteome