Comparative Study of [Three] LC−MALDI Workflows for the Analysis of Complex Proteomic Samples

• Published in: Journal of proteome research 2005-11, Vol.4 (6), p.1931-1941
• Main Authors: Hattan, Stephen J, Marchese, Jason, Khainovski, Nikita, Martin, Steve, Juhasz, Peter
• Format: Article
• Language: English
• Subjects: Cations; Chromatography; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Electrophoresis, Polyacrylamide Gel; Fungal Proteins - analysis; Mass Spectrometry; Peptides - chemistry; Proteomics - instrumentation; Proteomics - methods; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - instrumentation; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods; Time Factors; Ultraviolet Rays; Yeasts - metabolism
• ISSN: 1535-3893; 1535-3907
• DOI: 10.1021/pr050099e

Large-scale proteomic analyses frequently rely on high-resolution peptide separation of digested protein mixtures in multiple dimensions to achieve accuracy in sample detection and sensitivity in dynamic range of coverage. This study was undertaken to demonstrate the feasibility of MALDI MS/MS with off-line coupling to HPLC for the analysis of whole cell lysates of wild-type yeast by three different workflows:  SCX-RPHPLC−MS/MS, high-pH SAX-RPHPLC−MS/MS and RP (protein)-SCX-RPHPLC−MS/MS. The purpose of these experiments was to demonstrate the effect of a workflow on the end results in terms of the number of proteins detected, the average peptide coverage of proteins, and the number of redundant peptide sequencing attempts. Using 60 μg of yeast lysate, minor differences were seen in the number of proteins detected by each method (800−1200). The most significant differences were observed in redundancy of MS/MS acquisitions. Keywords: yeast • two dimensional chromatography • LC−MALDI • MALDI • workflow • proteomics • shotgun proteomics