Repeatability of peptide identifications in shotgun proteome analysis employing off-line two-dimensional chromatographic separations and ion-trap MS.
The repeatability of peptide identifications in shotgun proteome analyses employing strong cation-exchange-xion-pair RP HPLC hyphenated to ESI MS/MS was compared to an alternative scheme, comprising high-pH RP chromatography combined with low-pH ion-pair RP chromatography. Equivalent results were obtained with both methods in proteome analysis of Corynebacterium glutamicum. From a total number of 1350 to 1850 peptides identified in triplicate analyses of five consecutive fractions chosen from the first-dimension separation, 41-45% of the peptides were identified three times, whereas 16-22 and 37-39% of the peptides were identified only twice or once, respectively. A comparison of the repeatability of peptide identifications from complex samples upon 1- or 2-D chromatographic separation revealed that an additional separation dimension decreases the repeatability by approximately 25%.
Department of Chemistry, Instrumental Analysis and Bioanalysis, Saarland University, Saarbrücken, Germany.
Nathanaël
N
Delmotte
Maria
M
Lasaosa
Andreas
A
Tholey
Elmar
E
Heinzle
Alain
A
van Dorsselaer
Christian G
CG
Huber
D001426:Bacterial Proteins / Q000032:analysis*; D002845:Chromatography / Q000295:instrumentation / Q000379:methods*; D048230:Corynebacterium glutamicum / Q000737:chemistry; D010455:Peptides / Q000032:analysis*; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000295:instrumentation / Q000379:methods; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000295:instrumentation / Q000379:methods*
D001426:Bacterial Proteins; D010455:Peptides; D020543:Proteome