Dataset Information

High-Throughput Analysis of Intact Human Proteins Using UVPD and HCD on an Orbitrap Mass Spectrometer.

ABSTRACT: The analysis of intact proteins (top-down strategy) by mass spectrometry has great potential to elucidate proteoform variation, including patterns of post-translational modifications (PTMs), which may not be discernible by analysis of peptides alone (bottom-up approach). To maximize sequence coverage and localization of PTMs, various fragmentation modes have been developed to produce fragment ions from deep within intact proteins. Ultraviolet photodissociation (UVPD) has recently been shown to produce high sequence coverage and PTM retention on a variety of proteins, with increasing evidence of efficacy on a chromatographic time scale. However, utilization of UVPD for high-throughput top-down analysis to date has been limited by bioinformatics. Here we detected 153 proteins and 489 proteoforms using UVPD and 271 proteins and 982 proteoforms using higher energy collisional dissociation (HCD) in a comparative analysis of HeLa whole-cell lysate by qualitative top-down proteomics. Of the total detected proteoforms, 286 overlapped between the UVPD and HCD data sets, with 68% of proteoforms having C scores greater than 40 for UVPD and 63% for HCD. The average sequence coverage (28 ± 20% for UVPD versus 17 ± 8% for HCD, p < 0.0001) was found to be higher for UVPD than HCD and with a trend toward improvement in q value for the UVPD data set. This study demonstrates the complementarity of UVPD and HCD for more extensive protein profiling and proteoform characterization.

SUBMITTER: Cleland TP

PROVIDER: S-EPMC5549616 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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Publications

High-Throughput Analysis of Intact Human Proteins Using UVPD and HCD on an Orbitrap Mass Spectrometer.

Cleland Timothy P TP DeHart Caroline J CJ Fellers Ryan T RT VanNispen Alexandra J AJ Greer Joseph B JB LeDuc Richard D RD Parker W Ryan WR Thomas Paul M PM Kelleher Neil L NL Brodbelt Jennifer S JS

Journal of proteome research 20170419 5

The analysis of intact proteins (top-down strategy) by mass spectrometry has great potential to elucidate proteoform variation, including patterns of post-translational modifications (PTMs), which may not be discernible by analysis of peptides alone (bottom-up approach). To maximize sequence coverage and localization of PTMs, various fragmentation modes have been developed to produce fragment ions from deep within intact proteins. Ultraviolet photodissociation (UVPD) has recently been shown to p ...[more]

PMID: 28412815

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Project description:There is an immediate need for improved methods to systematically and precisely quantify large sets of peptides in complex biological samples. To date protein quantification in biological samples has been routinely performed on triple quadrupole instruments operated in selected reaction monitoring mode (SRM), and two major challenges remain. Firstly, the number of peptides to be included in one survey experiment needs to be increased to routinely reach several hundreds, and secondly, the degree of selectivity should be improved so as to reliably discriminate the targeted analytes from background interferences. High resolution and accurate mass (HR/AM) analysis on the recently developed Q-Exactive mass spectrometer can potentially address these issues. This instrument presents a unique configuration: it is constituted of an orbitrap mass analyzer equipped with a quadrupole mass filter as the front-end for precursor ion mass selection. This configuration enables new quantitative methods based on HR/AM measurements, including targeted analysis in MS mode (single ion monitoring) and in MS/MS mode (parallel reaction monitoring). The ability of the quadrupole to select a restricted m/z range allows one to overcome the dynamic range limitations associated with trapping devices, and the MS/MS mode provides an additional stage of selectivity. When applied to targeted protein quantification in urine samples and benchmarked with the reference SRM technique, the quadrupole-orbitrap instrument exhibits similar or better performance in terms of selectivity, dynamic range, and sensitivity. This high performance is further enhanced by leveraging the multiplexing capability of the instrument to design novel acquisition methods and apply them to large targeted proteomic studies for the first time, as demonstrated on 770 tryptic yeast peptides analyzed in one 60-min experiment. The increased quality of quadrupole-orbitrap data has the potential to improve existing protein quantification methods in complex samples and address the pressing demand of systems biology or biomarker evaluation studies.

Dataset Information

High-Throughput Analysis of Intact Human Proteins Using UVPD and HCD on an Orbitrap Mass Spectrometer.

Publications

High-Throughput Analysis of Intact Human Proteins Using UVPD and HCD on an Orbitrap Mass Spectrometer.

Similar Datasets

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