Utility of electrophoretically derived protein mass estimates as additional constraints in proteome analysis of human serum based on MS/MS analysis.
The proteome of a HUPO human serum reference sample was analyzed using multidimensional separation techniques at both the protein and the peptide levels. To eliminate false-positive identifications from the search results, we employed a data filtering method using molecular weight (MW) correlations derived from denaturing 1-DE. First, the six most abundant serum proteins were removed from the sample using immunoaffinity chromatography. 1-DE was then used to fractionate the remaining serum proteins according to the MW. Gel bands were isolated and in-gel digested with trypsin, and the resulting peptides were analyzed by 2-D LC/ESI-MS/MS. A SEQUEST search using the MS/MS results identified 494 proteins. Of these, 202 were excluded formally using protein data filtering as they were single-assignment proteins and their theoretical and electrophoretically-derived MWs did not correlate at high confidence. To evaluate this method, the results were compared with those of 1-D LC/MALDI-TOF/TOF and HUPO Plasma Proteome Project analyses. Our data filtering approach proved valuable in analysis of complex, large-scale proteomes such as human serum.
Kim
Jin Young
JY
Proteome Analysis Team, Korea Basic Science Institute, Yusung-Ku, Daejeon, Korea.
Lee
Jeong Hwa
JH
Park
Gun Wook
GW
Cho
Kun
K
Kwon
Kyung-Hoon
KH
Park
Young Mok
YM
Cho
Sang Yun
SY
Paik
Young-Ki
YK
Yoo
Jong Shin
JS
D000595:Amino Acid Sequence; D001798:Blood Proteins / Q000737:chemistry* / Q000302:isolation & purification; D002846:Chromatography, Affinity; D002853:Chromatography, Liquid; D030562:Databases, Protein; D004586:Electrophoresis / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010455:Peptides / Q000737:chemistry; D011506:Proteins / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D012015:Reference Standards; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013223:Statistics as Topic; D014357:Trypsin / Q000494:pharmacology
D001798:Blood Proteins; D010455:Peptides; D011506:Proteins; D014357:Trypsin