Multi-dimensional HPLC/MS of the nucleolar proteome using HPLC-chip/MS.
The proteome of the human nucleolus was investigated in a single analysis using off-line strong cation exchange chromatography and microfraction collection combined with HPLC-chip/MS. The analysis was conducted either as a 1-D workflow with HPLC-chip alone or as a 2-D workflow. Two hundred and six unique proteins were identified in the International Protein Index human database corresponding to 2024 unique tryptic peptides identified in the 2-D analysis. In contrast, only 34 proteins and 151 corresponding tryptic peptides were found by applying a 1-D separation strategy. This clearly indicated that the complexity of the samples required the combination of more than one orthogonal separation technique. Stringent database search criteria, including reversal of sequences and therefore better exclusion of false-positive identifications, were applied for reliable protein identification.
Vollmer
Martin
M
Agilent Technologies, Waldbronn, Germany. martin_vollmer@agilent.com
Hörth
Patric
P
Rozing
Gerard
G
Couté
Yohann
Y
Grimm
Rudi
R
Hochstrasser
Denis
D
Sanchez
Jean-Charles
JC
D002466:Cell Nucleolus / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D002852:Chromatography, Ion Exchange / Q000295:instrumentation / Q000379:methods*; D030562:Databases, Protein; D006801:Humans; D013058:Mass Spectrometry / Q000295:instrumentation / Q000379:methods*; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation / Q000379:methods*; D008969:Molecular Sequence Data; D020543:Proteome / Q000032:analysis*
D020543:Proteome