title: Evaluation of multidimensional chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) for large-scale protein analysis: the yeast proteome.
abstract: 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.
authors.affiliation: Department of Cell Biology, and Taplin Biological Mass Spectrometry Facility, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA.
authors.forename: Junmin
authors.initials: J
authors.lastname: Peng
authors.forename: Joshua E
authors.initials: JE
authors.lastname: Elias
authors.forename: Carson C
authors.initials: CC
authors.lastname: Thoreen
authors.forename: Larry J
authors.initials: LJ
authors.lastname: Licklider
authors.forename: Steven P
authors.initials: SP
authors.lastname: Gygi
mesh_terms: 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
chemical_list: D002412:Cations; D005656:Fungal Proteins; D007477:Ions; D010455:Peptides; D020543:Proteome