Lysine Propionylation To Boost Sequence Coverage and Enable a “Silent SILAC” Strategy for Relative Protein Quantification

Quantification in proteomics largely relies on the incorporation of stable isotopes, with protocols that either introduce the label through metabolic incorporation or chemical tagging. Most methods rely on the use of trypsin and/or LysC to generate labeled peptides. Although alternative proteases can enhance proteome coverage, generic quantitative methods that port over to such enzymes are lacking. Here we describe a quantification strategy amenable to most proteases, which involves propionylation of metabolically labeled lysine, using a “silent stable isotope labeling by amino acids in cell culture (SILAC)” strategy that reveals isotopic labels on second-stage mass spectrometry (MS2) fragmentation in a tandem mass tag (TMT)-like manner. We selectively propionylated lysine residues prior to digestion to generate pure ArgC-like digestion for trypsin and novel ArgN-like digestions for LysargiNase, by restricting digestion at lysine. The modification offers highly complementary sequence coverage, and even enhanced protein identification rates in certain situations (GluC digestion). Propionylated lysine residues were present in the majority of identified peptides generated from digests of cell lysates and led to the consistent release of an intense cyclic imine reporter ion at mass-to-charge ratio (<i>m</i>/<i>z</i>) 140 using higher-energy collisional dissociation. We grew A549 cells in media containing either l-1-¹³C-lysine or l-6-¹³C-lysine, to generate proteins that share the same accurate mass when paired. Peptides were indistinguishable on the first-stage mass spectrometry (MS1) level and, upon fragmentation, released reporter ions at <i>m</i>/<i>z</i> 140 and <i>m</i>/<i>z</i> 141, without otherwise affecting sequence ion mass. The quantification approach is independent of the number of peptide lysines and offers a new strategy for quantitative proteomics

Simultaneous Proteoform Analysis of Histones H3 and H4 with a Simplified Middle-Down Proteomics Method

Dynamic post-translational modifications of histones regulate transcriptional gene expression in eukaryotes. Unique combinations of modifications, almost exclusively displayed at the flexible N-terminal tails on histones, create distributions of proteoforms that need to be characterized in order to understand the complexity of gene regulation and how aberrant modification patterns influence disease. Although mass spectrometry is a preferred method for the analysis of histone modifications, information is lost when using conventional trypsin-based histone methods. Newer “middle-down” protocols may retain a greater fraction of the full proteoform distribution. We describe a strategy for the simultaneous characterization of histones H3 and H4 with near-complete retention of proteoform distributions, using a conventional proteomics liquid chromatography–tandem mass spectrometry (LC-MS/MS) configuration. The selective prolyl endoprotease neprosin generates convenient peptide lengths for retention and dispersion of modified H3 and H4 peptides on reversed-phase chromatography, offering an alternative to the hydrophilic interaction liquid chromatography typically used in middle-down methods. No chemical derivatizations are required, presenting a significant advantage over the trypsin-based protocol. Over 200 proteoforms can be readily profiled in a single analysis of histones from HeLa S3 cells. An in-gel digestion protocol provides additional options for effective histone analysis

High-Resolution Mapping of Carbene-Based Protein Footprints

Carbene chemistry has been used recently in structural mass spectrometry as a labeling method for mapping protein surfaces. The current study presents a method for quantitating label distribution at the amino acid level and explores the nature and basis for an earlier observation of labeling bias. With the use of a method based on liquid chromatography–tandem mass spectrometry (LC–MS/MS) applied to digests of holo-calmodulin, we developed a quantitation strategy to map site-specific incorporation of carbene, generated from photolysis of ionic label precursors 2-amino-4,4-azipentanoic acid and 4,4-azipentanoic acid. The approach provides reliable incorporation data for fragments generated by electron-transfer dissociation, whereas high-energy collisional dissociation leads to energy and sequence-dependent loss of the label as a neutral. However, both can produce data suitable for mapping residues in the interaction of holo-calmodulin with M13 peptide ligand. Site-specific labeling was monitored as a function of reagent, ionic strength, and temperature, demonstrating that electrostatic interactions at the protein surface can “steer” the distribution of label precursors to sites of surface charge and favor label insertion into residues in the vicinity of the surface charge. A further preference for insertion into carboxylates was observed, based on chemical reactivity. We suggest that decoupling surface partitioning from the chemistry of insertion offers a flexible, tunable labeling strategy for structural mass spectrometry that can be applied to a broad range of protein surface compositions and promotes the design of reagents to simplify the workflow

Assembly of Ebola Virus Matrix Protein VP40 Is Regulated by Latch-Like Properties of N and C Terminal Tails

<div><p>The matrix protein VP40 coordinates numerous functions in the viral life cycle of the Ebola virus. These range from the regulation of viral transcription to morphogenesis, packaging and budding of mature virions. Similar to the matrix proteins of other nonsegmented, negative-strand RNA viruses, VP40 proceeds through intermediate states of assembly (e.g. octamers) but it remains unclear how these intermediates are coordinated with the various stages of the life cycle. In this study, we investigate the molecular basis of synchronization as governed by VP40. Hydrogen/deuterium exchange mass spectrometry was used to follow induced structural and conformational changes in VP40. Together with computational modeling, we demonstrate that both extreme N and C terminal tail regions stabilize the monomeric state through a direct association. The tails appear to function as a latch, released upon a specific molecular trigger such as RNA ligation. We propose that triggered release of the tails permits the coordination of late-stage events in the viral life cycle, at the inner membrane of the host cell. Specifically, N-tail release exposes the L-domain motifs PTAP/PPEY to the transport and budding complexes, whereas triggered C-tail release could improve association with the site of budding.</p> </div

Carnivorous Nutrition in Pitcher Plants (<i>Nepenthes</i> spp.) via an Unusual Complement of Endogenous Enzymes

Plants belonging to the genus <i>Nepenthes</i> are carnivorous, using specialized pitfall traps called “pitchers” that attract, capture, and digest insects as a primary source of nutrients. We have used RNA sequencing to generate a cDNA library from the <i>Nepenthes</i> pitchers and applied it to mass spectrometry-based identification of the enzymes secreted into the pitcher fluid using a nonspecific digestion strategy superior to trypsin in this application. This first complete catalog of the pitcher fluid subproteome includes enzymes across a variety of functional classes. The most abundant proteins present in the secreted fluid are proteases, nucleases, peroxidases, chitinases, a phosphatase, and a glucanase. Nitrogen recovery involves a particularly rich complement of proteases. In addition to the two expected aspartic proteases, we discovered three novel nepenthensins, two prolyl endopeptidases that we name neprosins, and a putative serine carboxypeptidase. Additional proteins identified are relevant to pathogen-defense and secretion mechanisms. The full complement of acid-stable enzymes discovered in this study suggests that carnivory in the genus <i>Nepenthes</i> can be sustained by plant-based mechanisms alone and does not absolutely require bacterial symbiosis

Influence of RNA binding on domain translocations.

<p>(A) Scatterplot of the H/DX-MS peptide data summarizing the effect of 4 M urea on the monomer, with (B) the corresponding sequence plot of the data. (C) Scatterplot of the H/DX-MS peptide data summarizing the effect of 5′UGA3′ addition to the urea-treated VP40 monomer, with (D) the corresponding sequence plot of the data. Significant destabilizations are noted in blue, stabilizations in red, and no observable change in green. Deuteration changes are presented in millimass units (mmu). Dashed lines demarcate regions of statistical significance (see Materials and Methods).</p

RNA-induced tail separation and assembly.

<p>Top structures: VP40 monomer model with full sets of H/DX data showing RNA-induced changes (left structure) and urea-induced changes (right structure). Boxes mark the N and C-tails that are denatured only upon RNA binding. Orientation and color scheme as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039978#pone-0039978-g001" target="_blank">Figure 1</a>. Bottom structures: octamer model in side and top views, with the H/DX data superimposed. Blue denotes deprotection and red protection. Grey regions are not represented in H/DX data, and yellow highlights the bound 5′UGA3′.</p

Aspartic Protease Nepenthesin‑1 as a Tool for Digestion in Hydrogen/Deuterium Exchange Mass Spectrometry

Hydrogen/deuterium exchange coupled to mass spectrometry (HXMS) utilizes enzymatic digestion of proteins to localize the information about altered exchange patterns in protein structure. The ability of the protease to produce small peptides and overlapping fragments and provide sufficient coverage of the protein sequence is essential for localizing regions of interest. Recently, it was shown that there is an interesting group of proteolytic enzymes from carnivorous pitcher plants of the genus <i>Nepenthes</i>. In this report, we describe successful immobilization and the use of one of these enzymes, nepenthesin-1, in HXMS workflow. In contrast to pepsin, it has different cleavage specificities, and despite its high inherent susceptibility to reducing and denaturing agents, it is very stable upon immobilization and withstands even high concentration of guanidine hydrochloride and reducing agents. We show that denaturing agents can alter digestion by reducing protease activity and/or substrate solubility, and additionally, they influence the trapping of proteolytic peptides onto the reversed phase resin

Stability analysis of monomeric VP40 at the protein level.

<p>(A) Effect of VP40 concentration on its aggregation status as determined with H/DX-MS. (B) Conformational stability of monomeric VP40 upon treatment with increasing urea concentration, also using H/DX-MS as a readout. Each datapoint represents the average of 3 replicates (±1 SD) using a composite of peptides as described in Materials and Methods.</p

Free energy diagram for a proposed model of VP40 assembly.

<p>Destabilization of the monomer with urea mimics a native state where interactions between VP40 and the inner membrane induce a population of conformers, some of which are permissive of RNA binding. Tail release is triggered by RNA binding, followed in a concerted fashion by assembly and tail-mediated events in the viral life-cycle.</p