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Fully Automated Multidimensional Reversed-Phase Liquid Chromatography with Tandem Anion/Cation Exchange Columns for Simultaneous Global Endogenous Tyrosine Nitration Detection, Integral Membrane Protein Characterization, and Quantitative Proteomics Mapping in Cerebral Infarcts Quan Quan, Samuel S.W. Szeto, Henry Chun Hin Law, Zaijun Zhang, Yuqiang Wang, and Ivan K Chu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b02619 • Publication Date (Web): 03 Sep 2015 Downloaded from http://pubs.acs.org on September 4, 2015
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Analytical Chemistry
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Fully
Automated
Multidimensional
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Chromatography with Tandem Anion/Cation Exchange Columns for
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Simultaneous Global Endogenous Tyrosine Nitration Detection, Integral
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Membrane Protein
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Mapping in Cerebral Infarcts
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Quan Quan1, Samuel S.W. Szeto1, Henry Chun Hin Law1, Zaijun Zhang2, Yuqiang Wang2 and
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Ivan K. Chu*,1
Characterization,
and
Reversed-Phase
Quantitative
Liquid
Proteomics
8 9 1
Department of Chemistry, The University of Hong Kong, Hong Kong, China
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2
Institute of New Drug Research and Guangdong Province Key Laboratory of
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Pharmacodynamic Constituents of Traditional Chinese Medicine, College of Pharmacy, Jinan
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University, Guangzhou, 510632, China
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*Corresponding authors
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Email:
[email protected] 21 22 23
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Abstract
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Protein tyrosine nitration (PTN) is a signature hallmark of radical-induced nitrative stress in a
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wide range of pathophysiological conditions, with naturally occurring abundances at
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substoichiometric levels. In this present study, a fully automated four-dimensional platform,
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consisting
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complementary—strong anion (SAX) and cation exchange (SCX)—chromatographic
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separation stages inserted in tandem, was implemented for the simultaneous mapping of
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endogenous nitrated tyrosine–containing peptides within the global proteomic context of a
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Macaca fascicularis cerebral ischemic stroke model. This integrated RP–SA(C)X–RP
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platform was initially benchmarked through proteomics analyses of Saccharomyces cerevisiae,
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revealing extended proteome and protein coverage. A total of 27,144 unique peptides from
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3684 non-redundant proteins (1% global FDR) was identified from M. fascicularis cerebral
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cortex tissue. The inclusion of the S(A/C)X columns contributed to the increased detection of
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acidic, hydrophilic, and hydrophobic peptide populations; these separation features enabled the
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concomitant identification of 127 endogenous nitrated peptides and 137 transmembrane
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domain–containing peptides corresponding to integral membrane proteins, without the need
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for specific targeted enrichment strategies. The enhanced diversity of the peptide inventory
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obtained from the RP–SA(C)X–RP platform also improved analytical confidence in Isobaric
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Tags for Relative and Absolute Quantification (iTRAQ)–based proteomics analyses.
of
high-/low-pH
reversed-phase
dimensions
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Analytical Chemistry
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Introduction
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Oxidative stress is thought to play a significant role in the etiology of a wide range of human
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disorders, including neurodegenerative diseases, ischemia reperfusion injuries, aging,
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cardiovascular diseases, and diabetes;1,2 nevertheless, the precise mechanisms of these
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processes remain unclear. Reactive oxygen and nitrogen species are crucial substrates for the
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oxidative modification of proteins, resulting in potential alterations in their structures and/or
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functions.3-5 Protein tyrosine nitration (PTN)—a hallmark of nitrative stress—is a stable and
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irreversible oxidative post-translational modification (PTM) that results from the prevalence of
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cellular ROS and RNS.3-5 PTN is mediated mainly by the peroxynitrite anion (ONOO–), a
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highly reactive oxidant that modifies various types of biomolecules, produced from the rapid
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reaction of nitric oxide (NO•) with superoxide (O2•–).5 Modification with an
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electron-withdrawing nitro group at the ortho position of the phenol ring substantially alters the
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surface bulk and pKa of the protein tyrosine residues.4 The resulting consequences of PTN
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include altered protein function, increased proteolytic degradation sensitivity, enhanced
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immunogenicity, and variations in tyrosine phosphorylation.3,4
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PTN occurs at very low stoichiometry, estimated to be 0.001% or less of all tyrosine residues;3
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only a minute fraction of the total peptide population will contain these modifications. PTN
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modification is believed to occur at diverse local sequences, but with no observed consensus
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sequence motifs the prediction of PTN sites is currently impossible.3,4 Thus, unambiguous
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PTN identification requires comprehensive protein and proteome analyses, encompassing the
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sequence of the nitrated site—a level of detail that is not compulsory for protein identification.
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The challenges are even more formidable when identifying and characterizing tyrosine-nitrated
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proteins, where every nitrated peptide is crowded out and biased against by unmodified
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peptides when competing for ionization and product ion acquisition because of the sheer
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complexity and variability of the proteomes being examined: there may be up to 105 different
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protein species with a dynamic range of concentrations of up to 1010.6,7 Therefore,
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simultaneous proteome-scale qualitative and quantitative mapping of nitrated proteins with
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pinpointing of the exact modified tyrosyl sites and associated unmodified protein networks in a
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large-scale, high-throughput manner will be extremely helpful for deciphering the functional
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consequences of PTN modifications within the global cellular context.
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At present, the major route towards PTN characterization uses a combination of
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two-dimensional gel electrophoresis (2DE), immunoblotting, in-gel digestion, and MS
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identification.4 This approach is, however, limited mainly to abundant and soluble proteins
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lacking extreme values of pI and, in most instances is incapable of defining the exact PTN
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site(s).4 The limitations of the 2DE approach for analyzing hydrophobic membrane proteins is
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an important consideration when attempting to characterize PTN events, because selective
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tyrosine nitration has been observed in several instances for membrane protein transmembrane
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domains.4,8,9
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immunoaffinity chromatography, have also been applied to PTN detection. Although both
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approaches have high affinity, due to the antigen–antibody interaction strength, their
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specificity is antibody-dependent, as most exhibit nonspecific binding. Immunoprecipitation is
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a low-throughput method that is less sensitive than typical Western blot analyses,4 thereby
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bottlenecking downstream characterization.10,11 In contrast, immunoaffinity chromatography
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provides significantly higher throughput, but requires extensive incubation periods to increase
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its sensitivity and harsh elution conditions to enhance its selectivity. This necessity can
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decrease the antibody column lifetime, limiting the robustness of this approach.12-13 Chemical
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derivatization of 3-nitrotyrosine, with the conversion of these residues into chemically
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modified peptides amenable for subsequent chromatographic affinity capture, has been applied
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as an alternative approach.14 Unfortunately, this technique has not dramatically increased the
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number of detected nitration sites because it is prone to contamination and artifactual
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modifications.4,15,16 Although these various targeted approaches have enabled the identification
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of PTN with varying degrees of success, they do not permit the high-throughput simultaneous
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characterization of the underlying proteome. The challenges facing PTN characterization by
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either approach are also highlighted by previous comprehensive proteomic studies aimed at
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characterizing the mouse brain nitroproteome. Sacksteder et al. could identify only 35
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endogenous nitrotyrosine-modified peptides out of ~55,000 uniquely identified peptides;17
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even under pretreatment of mouse brain homogenate with a high dosage of peroxynitrite only
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102 and 150 enriched nitrated proteins and peptides, respectively, were identified.16
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The continuing evolution of multidimensional liquid chromatography/mass spectrometry
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(MDLC–MS)–based proteomics is facilitating the ongoing quest to unravel biological
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complexities, including protein variants formed through PTMs and protein isoforms. Recent
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MS advances have led to highly sensitive and rapid acquisition technologies for large-scale
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qualitative proteomics applications, allowing high-throughput identification of even those very
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low copy number peptides in data-dependent acquisition experiments.18,19 Nevertheless,
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technical challenges remain when attempting to cope with yet-to-be-sampled peptides and for
Techniques
involving
antibodies,
such
as
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PTMs that provide poor MS/MS signals and lower confidence in peptide identification. These
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situations are the result of poor electrospray ionization, due to a considerable degree of ion
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suppression or poor peptide fragmentation, obstructing peptide sequencing and PTM site
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localization.20 Electron capture/transfer dissociation is an emerging alternative analytical
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technique for PTM identification, isomeric residue differentiation [e.g., (iso)leucine], and de
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novo sequencing in proteomics analyses, but it is not applicable to molecules possessing high
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electron affinity (e.g., nitrated proteins).21,22 Thus, PTN identification is still performed
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routinely using low-energy tandem MS/MS in combination with high-resolution LC and
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database searching. Therefore, additional fractionation steps remain mandatory to ensure the
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recovery of a fraction of the under-sampled peptides and, thereby, extend the protein and
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proteome coverage.
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Recent advances in MDLC technology and its variety of combinations have led to
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multiplicative enhancements in the system orthogonality and peak capacity,23 aiming to
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minimize sample complexity, widen the overall dynamic range, and, consequently, increase the
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proteome coverage, as well as facilitate more comprehensive analyses of PTMs and isoform
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peptides.
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reversed-phase/strong anion exchange column chemistries (RP–SAX–RP) have been
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developed for large-scale qualitative and quantitative proteomics applications.24,25 The
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incompatibility of the mobile phase between the two RP dimensions was been resolved partly
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by inserting the SAX column between them. Anionic peptides that elute from the 1st-dimension
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high-pH RP column are directly applicable for SAX enrichment, thereby simplifying practical
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implementation. With any combination of separation chemistries, there will, however, be a
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portion of the peptide population that is not retained or fractionated efficiently. The
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aforementioned approach is amenable mainly to acidic peptides, because the SAX column
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material will retain and capture deprotonated peptide anions efficiently. However, considering
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that ~29% of human tryptic peptides have, theoretically, a neutral or basic net charge at a pH
