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What is the Impact of Phosphorylation on the Mass Spectrometry Quantification of Intact Phosphoproteins? Zhijie Wu, Timothy Neil Tiambeng, Wenxuan Cai, Bifan Chen, Ziqing Lin, Zachery R. Gregorich, and Ying Ge Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b05246 • Publication Date (Web): 22 Mar 2018 Downloaded from http://pubs.acs.org on March 23, 2018
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Analytical Chemistry
What Is the Impact of Phosphorylation on the Mass Spectrometry Quantification of Intact Phosphoproteins?
Zhijie Wu†, Timothy N. Tiambeng†, Wenxuan Cai‡§, Bifan Chen†, Ziqing Lin‡∥, Zachery R. Gregorich‡§, Ying Ge†‡∥
†
Department of Chemistry, ‡Department of Cell and Regenerative Biology, §Molecular and
Cellular Pharmacology Training Program, and ∥Human Proteomics Program, University of Wisconsin−Madison, Madison, Wisconsin 53705, United States
*To whom correspondence may be addressed: Dr. Ying Ge, Wisconsin Institute for Medical Research, Room 8551, 1111 Highland Ave, Madison, Wisconsin 53705, USA. E-mail:
[email protected]; Tel: 608-265-4744; Fax: 608-265-5512.
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Abstract Protein phosphorylation is a ubiquitous and critical post-translational modification (PTM) involved in numerous cellular processes. Mass spectrometry (MS)-based proteomics has emerged as the preferred technology for protein identification, characterization, and quantification. Whereas ionization/detection efficiency of peptides in electrospray (ESI)-MS are markedly influenced by the presence of phosphorylation, the physicochemical properties of intact proteins are assumed not to vary significantly due to the relatively smaller modification on large intact proteins. Thus the ionization/detection efficiency of intact phosphoprotein is hypothesized not to alter appreciably for subsequent MS quantification. However, this hypothesis has never been rigorously tested. Herein, we systematically investigated the impact of phosphorylation on ESI-MS quantification of mono- and multiply-phosphorylated proteins. We verified that a single phosphorylation did not appreciably affect the ESI-MS quantification of phosphoproteins as demonstrated in the enigma homolog isoform 2 (28 kDa) with mono-phosphorylation. Moreover, different ionization and desolvation parameters did not impact phosphoprotein quantification. In contrast to monophosphorylation,
multi-phosphorylation
phosphoproteins
likely
due
to
noticeably
differential
affected
ESI-MS
ionization/detection
quantification
efficiency
of
between
unphosphorylated and phosphorylated proteoforms as shown in the pentakis-phosphorylated βcasein (24 kDa).
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Analytical Chemistry
Protein phosphorylation is an important post-translational modification (PTM) that is involved in many critical cellular processes, including cell cycle control, cell growth, apoptosis, and signaling transduction pathways.1,2 Not surprisingly, altered phosphorylation levels have been associated with the development of diseases such as cardiovascular disease, cancer, and neurodegenerative disease.3-6 Moreover, recent evidence indicates that protein phosphorylation may also be useful as potential disease biomarkers.7 Therefore, accurate quantification of protein phosphorylation in different biological states not only can help elucidate intracellular signaling pathways that regulate various cellular processes, but may also be useful in understanding disease mechanism and diagnosis.8,9
Mass spectrometry (MS)-based proteomics has emerged as the preferred method for phosphoprotein identification, characterization, and quantification.10-12 The bottom-up MS-based phosphoproteomics approach, which commonly utilizes proteases to digest phosphoproteins into smaller peptides, is a high throughput method for quantification of phosphoproteins.13,14 However, ionization/detection efficiency of peptides in electrospray (ESI)-MS are markedly influenced by the presence of phosphorylation.15 Recently, top-down MS-based proteomics has emerged as the foremost method for the identification and quantification of proteoforms, a term adopted to represent the myriad protein products of a single gene generated via sequence variations (as a consequence of mutations/polymorphisms and/or alternative splicing), as well as post-translational modifications (PTMs).16 In top-down MS, intact proteins are analyzed, providing a “bird’s eye” view of all observed proteoforms in a given sample.17,18 Moreover, as the physicochemical properties of intact proteins are believed to be less impacted than peptides by the addition of smaller PTMs (e.g., phosphorylation), it is hypothesized that the effect of the small modifications on the ionization/detection efficiency of intact proteins will be negligible. Thus, top-down MS has 3 ACS Paragon Plus Environment
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been routinely employed for the quantification of modified and un-modified protein species in biological samples.3,19,20
In support of the long-held belief that the ionization/detection efficiency of intact proteins is not significantly impacted by PTMs, Kelleher and co-workers have demonstrated that the difference in ionization/detection efficiency between un-modified and acetylated intact recombinant H4 protein was minimal as observed by the small deviation (
