Enhanced Characterization of Complex Proteomic Samples Using LC−MALDI MS/MS:  Exclusion of Redundant Peptides from MS/MS Analysis in Replicate Runs

Abstract

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.

Tryptic Peptide Analysis of Ceruloplasmin in Dried Blood Spots Using Liquid Chromatography–Tandem Mass Spectrometry: Application to Newborn Screening

“…We developed a 7-min method without decreasing peak intensity of P-1 and P-1-IS, and further reduction of run time can be achieved by using a multicolumn LC system to carry out the separation of multiple samples simultaneously (16 ). The results from 8 DBS samples obtained from 7 WD patients and 1 carrier quantified using the LC-MS/MS method showed similar CP concentrations to those previously obtained using sandwich ELISA.…”

Comparison of 1‐D and 2‐D LC MS/MS methods for proteomic analysis of human serum

“…The overlap between the 2-D LC experiments indicates that the analytical methods are not complementary to a degree as suggested in literature [3,12,16]. Significant variability in peptide/protein identification is inherent to the DDA MS/MS method [28,53,54] and can be mistakenly interpreted as complementarities between different separation methods. The data-independent analysis used in this work has been shown to provide highly repeatable and reproducible results [55,56], although some variability remains for peptides approaching the detection (identification) limit.…”

An Enhanced Mass Spectrometry Approach Reveals Human Embryonic Stem Cell Growth Factors in Culture

“…To illustrate IE analysis accessing species of lower spectral abundance each entry was highlighted in the round in which it was identified with two or more peptides. In an effort to increase the total coverage, MS-based exclusion has been described in previously LC-MALDI experiments using a single repeat analysis (32,33). With LC-ESI-MS platforms, more extensive exclusion experiments have recently been reported, but these studies are based only on Based on combined analysis of replicates (H1 and H9 hESCs) utilizing all prefractionation approaches, all factors were identified with Ͼ2 unique peptides and were observed in both biological replicates.…”