Enhanced characterization of complex proteomic samples using LC-MALDI MS/MS: exclusion of redundant peptides from MS/MS analysis in replicate runs.
Due to the complexity of proteome samples, only a portion of peptides and thus proteins can be identified in a single LC-MS/MS analysis in current shotgun proteomics methodologies. It has been shown that replicate runs can be used to improve the comprehensiveness of the proteome analysis; however, high-intensity peptides tend to be analyzed repeatedly in different runs, thus reducing the chance of identifying low-intensity peptides. In contrast to commonly used online ESI-MS, offline MALDI decouples the separation from MS acquisition, thus allowing in-depth selection for specific precursor ions. Accordingly, we extended a strategy for offline LC-MALDI MS/MS analysis using a precursor ion exclusion list consisting of all identified peptides in preceding runs. The exclusion list eliminated redundant MS/MS acquisitions in subsequent runs, thus reducing MALDI sample depletion and allowing identification of a larger number of peptide identifications in the cumulative dataset. In the analysis of the digest of an Escherichia coli lysate, the exclusion list strategy resulted in a 25% increase in the number of unique peptide identifications in the second run, in contrast to simply pooling MS/MS data from two replicate runs. To reduce the increased LC analysis time for repeat runs, a four-column multiplexed LC system was developed to carry out separation simultaneously. The multiplexed LC-MALDI MS provides a high-throughput platform to utilize the exclusion list strategy in proteome analysis.
Chen
Hsuan-shen
HS
Barnett Institute and Department of Chemistry and Chemical Biology, Northeastern University, Boston, Massachusetts 02115, USA.
Rejtar
Tomas
T
Andreev
Victor
V
Moskovets
Eugene
E
Karger
Barry L
BL
D000595:Amino Acid Sequence; D002853:Chromatography, Liquid / Q000379:methods*; D029968:Escherichia coli Proteins / Q000737:chemistry; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis* / Q000737:chemistry*; D020543:Proteome / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
D029968:Escherichia coli Proteins; D010455:Peptides; D020543:Proteome