Post Analysis Data Acquisition for the Iterative MS/MS Sampling of Proteomics Mixtures

Abstract

The identification of peptides by microcapillary liquid chromatography-tandem mass spectrometry (µLC-MS/MS) has become routine because of the development of fast scanning mass spectrometers, data-dependent acquisition, and database searching algorithms. However, many peptides within the detection limit of the mass spectrometer remain unidentified because of limitations in MS/MS sampling speed despite the dynamic range and peak capacity of the instrument. We have developed an automated approach that uses the mass spectra from high resolution µLC-MS data to define the molecular species present in the mixture and directs the acquisition of MS/MS spectra to precursors that were missed in prior analyses. This approach increases the coverage of the molecular species sampled by MS/MS and consequently the number of peptides and proteins identified during the acquisition of technical or biological replicates using a simple one-dimensional chromatographic separation. The combination of a unique workflow and custom software contribute to the improved identification of molecular features detected in proteomics experiments of complex protein mixtures.

Proteomics enhances evolutionary and functional analysis of reproductive proteins

“…(33) We anticipate that as MS technologies advance, differential labeling approaches will grow more powerful. New methods that specifically direct the mass spectrometer to analyze low-abundance proteins (45,46) will increase the sensitivity of detection for transferred proteins, and within several years, MS instruments will be able to discern in real time which peptides are unlabeled (representing transferred male proteins) and which are labeled (representing femalederived proteins). Such an advance would further increase the sensitivity to detect male-derived proteins, but it would also enable the identification of female-derived proteins that were newly synthesized upon mating by allowing researchers to compare which 15 N peptides were identified in mated versus virgin female samples.…”

Automated Precursor Ion Exclusion During LC-MS/MS Data Acquisition for Optimal Ion Identification

“…Background ions as well as precursor ions that have been selected for MS/MS in a previous run will be put into the exclusion list and, therefore, will not be selected for MS/MS in later runs. Although using an exclusion list was not favored by Hoopmann et al attributable to the large size of the list [10], it served us well in most of our applications. This report describes in details how an exclusion list is generated, with a purpose of maximizing both the quality and number of unique MS/MS acquired during an LC-MS/MS experiment.…”

“…An automatic ISDDA technology has been developed by Hoopmann et al for post-analysis data acquisition (PAnDA), when a precursor ion list is used instead of an exclusion list to select the precursor ions intelligently for the next LC-MS/ MS run [10]. Using a precursor ion list offers a great advantage when a sample is analyzed multiple times, such as in many experimental settings when technical replicates are required.…”

Software Analysis of Uncorrelated MS1 Peaks for Discovery of Post-Translational Modifications

“…Another method has been described in which software builds a database of algorithmically selected peaks and directly interfaces with the instrument acquisition process. In this manner, it is able to generate inclusion lists using different peaks from previous runs, effectively increasing MS/MS sequence coverage and the number of proteins identified, while accomplishing this in a fully automated manner [55]. Despite improvements in instrumentation, techniques, and efforts in data analysis, we are still falling short of identifying all peptides and their isoforms in a sample, particularly in complex mixtures.…”