Exploiting the complementary nature of LC/MALDI/MS/MS and LC/ESI/MS/MS for increased proteome coverage.
The goal of this work was to evaluate the improvement in proteome coverage of complex protein mixtures gained by analyzing samples using both LC/ESI/MS/MS and LC/MALDI/MS/MS. Parallel analyses of a single sample were accomplished by interfacing a Probot fractionation system with a nanoscale LC system. The Probot was configured to perform a post-column split such that a fraction (20%) of the column effluent was sent for on-line LC/ESI/MS/MS data acquisition, and the majority of the sample (80%) was mixed with a matrix solution and deposited onto the MALDI target plate. The split-flow approach takes advantage of the concentration sensitive nature of ESI and provides sufficient quantity of sample for MALDI/MS/MS. Hybrid quadrupole time-of-flight mass spectrometers were used to acquire LC/ESI/MS/MS data and LC/MALDI/MS/MS data from a tryptic digest of a preparation of mammalian mitochondrial ribosomes. The mass spectrometers were configured to operate in a data dependent acquisition mode in which precursor ions observed in MS survey scans are automatically selected for interrogation by MS/MS. This type of acquisition scheme maximizes the number of peptide fragmentation spectra obtained and is commonly referred to as shotgun analysis. While a significant degree of overlap (63%) was observed between the proteins identified in the LC/ESI/MS/MS and LC/MALDI/MS/MS data sets, both unique peptides and unique proteins were observed by each method. These results demonstrate that improved proteome coverage can be obtained using a combination of these ionization techniques.
Bodnar
Wanda M
WM
Proteomic Technologies, Genetic Research Division, GlaxoSmithKline, Research Triangle Park, North Carolina 27709-3398, USA. wanda.m.bodnar@gsk.com
Blackburn
R Kevin
RK
Krise
Jo M
JM
Moseley
M Arthur
MA
D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid / Q000379:methods; D008928:Mitochondria / Q000737:chemistry; D020543:Proteome / Q000032:analysis* / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D012269:Ribosomal Proteins / Q000032:analysis / Q000737:chemistry; D012270:Ribosomes / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D020543:Proteome; D012269:Ribosomal Proteins