High-throughput mass spectrometric discovery of protein post-translational modifications.

The availability of genome sequences, affordable mass spectrometers and high-resolution two-dimensional gels has made possible the identification of hundreds of proteins from many organisms by peptide mass fingerprinting. However, little attention has been paid to how information generated by these means can be utilised for detailed protein characterisation. Here we present an approach for the systematic characterisation of proteins using mass spectrometry and a software tool FindMod. This tool, available on the internet at http://www.expasy.ch/sprot/findmod.html , examines peptide mass fingerprinting data for mass differences between empirical and theoretical peptides. Where mass differences correspond to a post-translational modification, intelligent rules are applied to predict the amino acids in the peptide, if any, that might carry the modification. FindMod rules were constructed by examining 5153 incidences of post-translational modifications documented in the SWISS-PROT database, and for the 22 post-translational modifications currently considered (acetylation, amidation, biotinylation, C-mannosylation, deamidation, flavinylation, farnesylation, formylation, geranyl-geranylation, gamma-carboxyglutamic acids, hydroxylation, lipoylation, methylation, myristoylation, N -acyl diglyceride (tripalmitate), O-GlcNAc, palmitoylation, phosphorylation, pyridoxal phosphate, phospho-pantetheine, pyrrolidone carboxylic acid, sulphation) a total of 29 different rules were made. These consider which amino acids can carry a modification, whether the modification occurs on N-terminal, C-terminal or internal amino acids, and the type of organisms on which the modification can be found. We illustrate the utility of the approach with proteins from 2-D gels of Escherichia coli and sheep wool, where post-translational modifications predicted by FindMod were confirmed by MALDI post-source decay peptide fragmentation. As the approach is amenable to automation, it presents a potentially large-scale means of protein characterisation in proteome projects.     

Global proteome discovery using an online three-dimensional LC-MS/MS

We have developed a proteomics technology featuring on-line three-dimensional liquid chromatography coupled to tandem mass spectrometry (3D LC-MS/MS). Using 3D LC-MS/MS, the yeast-soluble, urea-solubilized peripheral membrane and SDS-solubilized membrane protein samples collectively yielded 3019 unique yeast protein identifications with an average of 5.5 peptides per protein from the 6300-gene Saccharomyces Genome Database searched with SEQUEST. A single run of the urea-solubilized sample yielded 2255 unique protein identifications, suggesting high peak capacity and resolving power of 3D LC-MS/MS. After precipitation of SDS from the digested membrane protein sample, 3D LC-MS/MS allowed the analysis of membrane proteins. Among 1221 proteins containing two or more predicted transmembrane domains, 495 such proteins were identified. The improved yeast proteome data allowed the mapping of many metabolic pathways and functional categories. The 3D LC-MS/MS technology provides a suitable tool for global proteome discovery.     

Immonium ion scanning for the discovery of post-translational modifications and its application to histones

This study investigates the use of immonium ion scanning for the discovery of methylated and acetylated peptides. Tandem mass spectrometry of modified and unmodified versions of identical peptides revealed ions of 98, 112 and 126 m/ z specifically in association with mono-, dimethylated and acetylated lysine, respectively. Ions of 143 m/ z were seen to be associated with monomethylated arginine, although were not unique to this amino acid. Use of immonium ion scanning with differing collision energies (35, 55, 75, 95, 115 eV) showed that where immonium ions are strong and unique for a modified amino acid, the discovery rate of modified peptides can be improved up to 4-fold over control analyses. The position of an amino acid in a peptide, being terminal or internal, also affected the efficiency of identification of modified peptides. Higher collision energy scanning was required for the most effective identification of peptides with internal modified residues. We conclude that immonium ion scanning, particularly with a range of collision energies, can improve the discovery efficiency of post-translational modifications in peptides.     

Roles of acetylation and other post-translational modifications in melanocortin function and interactions with endorphins

Phylogenetic, developmental, anatomic, and stimulus-specific variations in post-translational processing of POMC are well established. For melanocortins, the role of alpha-N-acetylation and the selective activities of alpha, beta, and gamma forms are of special interest. Acetylation may shift the predominant activity of POMC products between endorphinergic and melanocortinergic actions-which are often in opposition. This review addresses: (1) variations in POMC processing; (2) the influence of acetylation on the functional activity of alpha-MSH; (3) state- and stimulus-dependent effects on the proportional distribution of forms of melanocortins and endorphins; (4) divergent effects of alpha-MSH and beta-endorphin administration; (5) potential roles of beta- and gamma-MSH.     

Mass spectrometry-based strategies for characterization of histones and their post-translational modifications

Due to the intimate interactions between histones and DNA, the characterization of histones has become the focus of great attention. A series of mass spectrometry-based technologies have been dedicated to the characterization and quantitation of different histone forms. This review focuses on the discussion of mass spectrometry-based strategies used for the characterization of histones and their post-translational modifications.     

Characterization of pertussis toxin by LC-MS/MS

Liquid chromatography-mass spectrometry (LC-MS) with a dual spray electrospray ionization source has been used to measure the molecular weights of pertussis toxin (PT) subunits. Measurement accuracy better than 0.4 Da was achieved for all PT subunits in the molecular weight range of 11,000 to 27,000 Da. At this mass assignment accuracy level, the sequences of the PT subunits investigated in this study are easily determined based on molecular weight alone. The subunits 1, 2, and 5 of PT were observed to undergo oxidation under normal storage conditions as ammonium sulfate suspension at 2 to 8 degrees C. These oxidized subunits can be separated completely or partially by reverse-phase high-performance liquid chromatography (HPLC) from their native counterparts. For the determination of oxidation sites, the oxidized subunits and their nonoxidized counterparts were fraction collected, trypsin digested, and mapped by LC-MS. The oxidized peptides and their nonoxidized counterparts were further studied by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to confirm their identities. The methionines at position 212 of subunit 1, at position 89 of subunit 2, and at position 40 of subunit 5 were found to be the primary sites of oxidation.     

Mass spectrometry-based strategies for characterization of histones and their post-translational modifications

Due to the intimate interactions between histones and DNA, the characterization of histones has become the focus of great attention. A series of mass spectrometry-based technologies have been dedicated to the characterization and quantitation of different histone forms. This review focuses on the discussion of mass spectrometry-based strategies used for the characterization of histones and their post-translational modifications.     

Regulation of pannexin channels by post-translational modifications

The large-pore channels formed by the pannexin family of proteins have been implicated in many physiological and pathophysiological functions, mainly through their ATP release function. However, a tight regulation of channel opening is necessary to modulate their function in vivo. Post-translational modifications have been postulated as some of the regulating mechanisms for Panx1, while Panx2 and Panx3 have not been as well characterized. Positive regulators include caspase cleavage to open Panx1 channels in apoptotic cells, and activation by Src family kinases via ionotropic receptors in neurons and macrophages. S-nitrosylation of cysteines has been shown to both inhibit and activate the Panx1 channel in different cell types. All three pannexins are N-glycosylated but to different levels of modification. Their diverse glycosylation appears to regulate cellular localization, intermixing, and may restrict their ability to function as inter-cellular channels. It is clear that our understanding of pannexin post-translational modification and their role in channel function regulation is still in its infancy even a decade after their discovery.     

The regulation of necroptosis by post-translational modifications

Necroptosis is a caspase-independent, lytic form of programmed cell death whose errant activation has been widely implicated in many pathologies. The pathway relies on the assembly of the apical protein kinases, RIPK1 and RIPK3, into a high molecular weight cytoplasmic complex, termed the necrosome, downstream of death receptor or pathogen detector ligation. The necrosome serves as a platform for RIPK3-mediated phosphorylation of the terminal effector, the MLKL pseudokinase, which induces its oligomerization, translocation to, and perturbation of, the plasma membrane to cause cell death. Over the past 10 years, knowledge of the post-translational modifications that govern RIPK1, RIPK3 and MLKL conformation, activity, interactions, stability and localization has rapidly expanded. Here, we review current knowledge of the functions of phosphorylation, ubiquitylation, GlcNAcylation, proteolytic cleavage, and disulfide bonding in regulating necroptotic signaling. Post-translational modifications serve a broad array of functions in modulating RIPK1 engagement in, or exclusion from, cell death signaling, whereas the bulk of identified RIPK3 and MLKL modifications promote their necroptotic functions. An enhanced understanding of the modifying enzymes that tune RIPK1, RIPK3, and MLKL necroptotic functions will prove valuable in efforts to therapeutically modulate necroptosis.Subject terms: Kinases, Proteomics, Cell biology     

Regulation of dynamin family proteins by post-translational modifications

Dynamin superfamily proteins comprising classical dynamins and related proteins are membrane remodelling agents involved in several biological processes such as endocytosis, maintenance of organelle morphology and viral resistance. These large GTPases couple GTP hydrolysis with membrane alterations such as fission, fusion or tubulation by undergoing repeated cycles of self-assembly/disassembly. The functions of these proteins are regulated by various post-translational modifications that affect their GTPase activity, multimerization or membrane association. Recently, several reports have demonstrated variety of such modifications providing a better understanding of the mechanisms by which dynamin proteins influence cellular responses to physiological and environmental cues. In this review, we discuss major post-translational modifications along with their roles in the mechanism of dynamin functions and implications in various cellular processes.     

Regulation of mammalian mitochondrial translation by post-translational modifications

Mitochondria are responsible for the production of over 90% of the energy in eukaryotes through oxidative phosphorylation performed by electron transfer and ATP synthase complexes. Mitochondrial translation machinery is responsible for the synthesis of 13 essential proteins of these complexes encoded by the mitochondrial genome. Emerging data suggest that acetyl-CoA, NAD(+), and ATP are involved in regulation of this machinery through post-translational modifications of its protein components. Recent high-throughput proteomics analyses and mapping studies have provided further evidence for phosphorylation and acetylation of ribosomal proteins and translation factors. Here, we will review our current knowledge related to these modifications and their possible role(s) in the regulation of mitochondrial protein synthesis using the homology between mitochondrial and bacterial translation machineries. However, we have yet to determine the effects of phosphorylation and acetylation of translation components in mammalian mitochondrial biogenesis. This article is part of a Special Issue entitled: Mitochondrial Gene Expression.     

Regulation of protease-activated receptor signaling by post-translational modifications

Protease-activated receptors (PARs) are a unique family of G-protein-coupled receptors (GPCRs) that are irreversibly activated following proteolytic cleavage of their extracellular N-terminus. PARs play critical functions in hemostasis, thrombosis, inflammation, embryonic development, and cancer progression. Because of the irreversible proteolytic nature of PAR activation, signaling by the receptors is tightly regulated. Three distinct processes including desensitization, internalization, and lysosomal degradation, regulate the temporal and spatial aspects of activated PAR signaling. Post-translational modifications play a critical role in regulating each of these processes and here we review the nature of PAR post-translational modifications and their importance in signal regulation. The PARs are activated by numerous proteases, and some can elicit distinct cellular responses, how this biased agonism is determined is unknown. Further study of the function of post-translational modifications of the PARs will lead to a greater understanding of the physiological regulation of baised agonism and how PAR signaling is precisely controlled in different cellular contexts.     

DeltAMT: a statistical algorithm for fast detection of protein modifications from LC-MS/MS data

Identification of proteins and their modifications via liquid chromatography-tandem mass spectrometry is an important task for the field of proteomics. However, because of the complexity of tandem mass spectra, the majority of the spectra cannot be identified. The presence of unanticipated protein modifications is among the major reasons for the low spectral identification rate. The conventional database search approach to protein identification has inherent difficulties in comprehensive detection of protein modifications. In recent years, increasing efforts have been devoted to developing unrestrictive approaches to modification identification, but they often suffer from their lack of speed. This paper presents a statistical algorithm named DeltAMT (Delta Accurate Mass and Time) for fast detection of abundant protein modifications from tandem mass spectra with high-accuracy precursor masses. The algorithm is based on the fact that the modified and unmodified versions of a peptide are usually present simultaneously in a sample and their spectra are correlated with each other in precursor masses and retention times. By representing each pair of spectra as a delta mass and time vector, bivariate Gaussian mixture models are used to detect modification-related spectral pairs. Unlike previous approaches to unrestrictive modification identification that mainly rely upon the fragment information and the mass dimension in liquid chromatography-tandem mass spectrometry, the proposed algorithm makes the most of precursor information. Thus, it is highly efficient while being accurate and sensitive. On two published data sets, the algorithm effectively detected various modifications and other interesting events, yielding deep insights into the data. Based on these discoveries, the spectral identification rates were significantly increased and many modified peptides were identified.     

Protein identification by syringe pump-driven reversed-phase LC-MS/MS

In typical mass spectrometry-based protein identification using peptide fragmentation fingerprinting, front-end separation plays a critical role in successful peptide sequencing. This separation step demands a great deal of time and usually is the rate-limiting step for the whole process. Here we provide an alternative separation method, based on a simple nanoflow delivery system, that is able to shorten the separation time considerably. This system consists of a 25-mul syringe connected to a manually packed reversed-phase mini-capillary column that can be directly coupled to an electrospray ionization tandem mass spectrometer. A syringe pump is then used to deliver the peptide mixtures at a nanoscale flow rate. We examined the efficiency and efficacy of this method by analyzing the tryptic peptides of bovine serum albumin and of 10 Escherichia coli proteins separated by two-dimensional gel electrophoresis (2DE). The results showed that identification of each protein could be achieved successfully within 25 min by using the disposable mini-capillary column. Moreover, all 2DE-separated E. coli proteins were identified at high confidence levels. Together, our data suggest that this method is a suitable option for mass spectrometry-based protein identification.     

Novel approach in LC-MS/MS using MRM to generate a full profile of acyl-CoAs: discovery of acyl-dephospho-CoAs

A metabolomic approach to selectively profile all acyl-CoAs was developed using a programmed multiple reaction monitoring (MRM) method in LC-MS/MS and was employed in the analysis of various rat organs. The programmed MRM method possessed 300 mass ion transitions with the mass difference of 507 between precursor ion (Q1) and product ion (Q3), and the precursor ion started from m/z 768 and progressively increased one mass unit at each step. Acyl-dephospho-CoAs resulting from the dephosphorylation of acyl-CoAs were identified by accurate MS and fragmentation. Acyl-dephospho-CoAs were also quantitatively scanned by the MRM method with the mass difference of 427 between Q1 and Q3 mass ions. Acyl-CoAs and dephospho-CoAs were assayed with limits of detection ranging from 2 to 133 nM. The accuracy of the method was demonstrated by assaying a range of concentrations of spiked acyl-CoAs with the results of 80–114%. The distribution of acyl-CoAs reflects the metabolic status of each organ. The physiological role of dephosphorylation of acyl-CoAs remains to be further characterized. The methodology described herein provides a novel strategy in metabolomic studies to quantitatively and qualitatively profile all potential acyl-CoAs and acyl-dephospho-CoAs.     

Improving LC-MS sensitivity through increases in chromatographic performance: comparisons of UPLC-ES/MS/MS to HPLC-ES/MS/MS

Recent technological advances have made available reverse phase chromatographic media with a 1.7 microm particle size along with a liquid handling system that can operate such columns at much higher pressures. This technology, termed ultra performance liquid chromatography (UPLC), offers significant theoretical advantages in resolution, speed, and sensitivity for analytical determinations, particularly when coupled with mass spectrometers capable of high-speed acquisitions. This paper explores the differences in LC-MS performance by conducting a side-by-side comparison of UPLC for several methods previously optimized for HPLC-based separation and quantification of multiple analytes with maximum throughput. In general, UPLC produced significant improvements in method sensitivity, speed, and resolution. Sensitivity increases with UPLC, which were found to be analyte-dependent, were as large as 10-fold and improvements in method speed were as large as 5-fold under conditions of comparable peak separations. Improvements in chromatographic resolution with UPLC were apparent from generally narrower peak widths and from a separation of diastereomers not possible using HPLC. Overall, the improvements in LC-MS method sensitivity, speed, and resolution provided by UPLC show that further advances can be made in analytical methodology to add significant value to hypothesis-driven research.