Characterization and Quantification of Intact 26S Proteasome Proteins by Real-Time Measurement of Intrinsic Fluorescence Prior to Top-down Mass Spectrometry

Jason D Russell,Joshua J Coon,Adam J Book,Mark Scalf,Richard D Vierstra,Lloyd M Smith,Daniel T Ladror,Sonja Hess

doi:10.1371/journal.pone.0058157

Jason D Russell, Joshua J Coon + Show 6 more

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https://doi.org/10.1371/journal.pone.0058157

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Abstract

Quantification of gas-phase intact protein ions by mass spectrometry (MS) is impeded by highly-variable ionization, ion transmission, and ion detection efficiencies. Therefore, quantification of proteins using MS-associated techniques is almost exclusively done after proteolysis where peptides serve as proxies for estimating protein abundance. Advances in instrumentation, protein separations, and informatics have made large-scale sequencing of intact proteins using top-down proteomics accessible to the proteomics community; yet quantification of proteins using a top-down workflow has largely been unaddressed. Here we describe a label-free approach to determine the abundance of intact proteins separated by nanoflow liquid chromatography prior to MS analysis by using solution-phase measurements of ultraviolet light-induced intrinsic fluorescence (UV-IF). UV-IF is measured directly at the electrospray interface just prior to the capillary exit where proteins containing at least one tryptophan residue are readily detected. UV-IF quantification was demonstrated using commercially available protein standards and provided more accurate and precise protein quantification than MS ion current. We evaluated the parallel use of UV-IF and top-down tandem MS for quantification and identification of protein subunits and associated proteins from an affinity-purified 26S proteasome sample from Arabidopsis thaliana. We identified 26 unique proteins and quantified 13 tryptophan-containing species. Our analyses discovered previously unidentified N-terminal processing of the β6 (PBF1) and β7 (PBG1) subunit - such processing of PBG1 may generate a heretofore unknown additional protease active site upon cleavage. In addition, our approach permitted the unambiguous identification and quantification both isoforms of the proteasome-associated protein DSS1.

Highlights

Protein analysis is complicated by sequence deviation from that predicted by the genome and the 200–300 known dynamic posttranslational modifications (PTMs) that occur [1,2,3]
Protein ultraviolet light-induced intrinsic fluorescence (UV-IF) response increased with increasing protein mass (Figure 1, upper right); while, quadrupole linear ion trap (QLT)-mass spectrometry (MS) and FT-MS response generally decreased with increasing protein mass
These observations agree with previous reports of UV-IF of intact proteins [48].Decreasing MS signals for both mass analyzers were observed for proteins of increasing mass and may be attributed to several factors including low ionization and transmission efficiencies, as well as diminished detector signal due to decreased ion-toelectron conversion efficiency (QLT) or ion cloud dephasing associated with gas-phase collisional cross sections (FT) [38,39,40,49,50,51,52,53]

Summary

Introduction

Protein analysis is complicated by sequence deviation from that predicted by the genome (e.g., single nucleotide polymorphisms, alternative splicing) and the 200–300 known dynamic posttranslational modifications (PTMs) that occur [1,2,3]. Intact protein analysis by top-down mass spectrometry (MS) enables the examination of combinatorial PTMs and the ability to identify splice variants while these biologically important features often remain veiled or ambiguous after proteolysis [4,5,6]. Recent work demonstrated that thousands of protein forms can be identified with state-of-the-art top-down technology allowing comprehensive proteome analysis at the intact protein level [23]. Protein quantification is becoming increasingly important because it is often the changes in protein expression and modification state that drive biological events, not exclusively the presence or absence of a particular protein or proteoform (proteoforms describe the protein products derived from a single gene where each uniquely modified protein is termed a ‘proteoform’) [24]

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