Exploiting the complementary nature of LC/MALDI/MS/MS and LC/ESI/MS/MS for increased proteome coverage

Abstract

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. (J Am Soc Mass Spectrom 2003, 14, 971-979)

Analysis of protein-protein interaction surfaces using a combination of efficient lysine acetylation and nanoLC-MALDI-MS/MS applied to the E9:Im9 bacteriotoxin—immunity protein complex

“…In recent years commercial LC-MALDI interfaces have become available. LC-MALDI can be used to obtain increased proteome coverage by applying the complementary combination of LC-ESI-MS/MS and LC-MALDI-MS/MS [24,25]. Related to this work, Li et al used LC-MALDI-MS/MS in combination with isotope labeling and limited enzymatic digestion to monitor conformational changes of cardiac troponin C induced by calcium binding [26].…”

Post-translational Modifications and Mass Spectrometry Detection

“…In addition, the different discrimination of MALDI and ESI peptides also contributes to different protein coverage rates. Several authors have reported different results when using these two ionization methods (22,37,(39)(40)(41). These results globally show that ESI allows a slightly better protein coverage rate.…”

Assessment of Proteomics Strategies for Plant Cell Wall Glycosyltransferases in Wheat, a Non-Model Species: Glucurono(Arabino)Xylan as a Case Study

“…Adapted from (http://synteny.cnr.berkeley.edu/wiki/index.php/Sequenced_plant_genomes) (MALDI) or ElectroSpray Ionization (ESI), (iii) mass spectrometry (MS) analysis of the ionized peptides (received from the source) by tandem mass spectrometry (MS/MS), also called collision-induced dissociation (CID). This step generally delivers large informationrich files of MS/MS spectra (Bodnar et al, 2003). The third and critical step concerns the use of these MS/MS spectra in the identification of the exact proteins or at least their closest homologous proteins from the databases.…”