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BBA - Proteins and Proteomics (v.1764, #12)

Editorial Board (pp. ii).

Special issue: Posttranslational modifications by Helmut E. Meyer; Michael Hamacher (pp. 1787-1787).

Analysis of posttranslational modifications exemplified using protein kinase A by Frank Gesellchen; Oliver Bertinetti; Friedrich W. Herberg (pp. 1788-1800).

With the completion of the major genome projects, one focus in biomedical research has shifted from the analysis of the rather static genome to the highly dynamic proteome. The sequencing of whole genomes did not lead to much anticipated insights into disease mechanisms; however, it paved the way for proteomics by providing the databases for protein identification by peptide mass fingerprints. The relative protein distribution within a cell or tissue is subject to change upon external and internal stimuli. Signal transduction events extend beyond a simple change in protein levels; rather they are governed by posttranslational modifications (PTMs), which provide a quick and efficient way to modulate cellular signals. Because most PTMs change the mass of a protein, they are amenable to analysis by mass spectrometry. Their investigation adds a level of functionality to proteomics, which can be expected to greatly aid in the understanding of the complex cellular machinery involved in signal transduction, metabolism, differentiation or in disease. This review provides an overview on posttranslational modifications exemplified on the model system cAMP-dependent protein kinase. Strategies for detection of selected PTMs are described and discussed in the context of protein kinase function.

Keywords: cAMP-dependent protein kinase; Phosphorylation; Myristoylation; Deamidation; Mass spectrometry

Protein processing and other modifications analyzed by diagonal peptide chromatography by Kris Gevaert; Petra Van Damme; Bart Ghesquière; Joël Vandekerckhove (pp. 1801-1810).

Diagonal peptide chromatography consists of two consecutive, identical peptide separations with in between an enzymatic or chemical alteration of the side-chain structure of selected peptides. Such selected and altered peptides acquire different chromatographic properties thereby segregating from non-altered peptides in a series of secondary peptide separations. Originally described by Brown and Hartley in 1966, we have modified the technique such that it can be used for higher throughput gel-free proteomics. Our technique is termed COmbined FRActional DIagonal Chromatography (COFRADIC) and exploits evoked differences of the hydrophobicity of peptides in reverse-phase liquid chromatography. One important advantage of COFRADIC is its versatility: by changing the alteration reaction, different classes of peptides are sorted and finally analyzed. We previously published protocols and applications for separating methionyl, cysteinyl, amino terminal and phosphorylated peptides. In this review, we assess the potential of COFRADIC for the analysis of several posttranslational modifications emphasizing on in vivo protein processing events. Additional modifications that can be analyzed include phosphorylation and N-glycosylation. The potential of COFRADIC for isolating peptides holding such modified amino acids are discussed here.

Keywords: Diagonal chromatography; Protein processing; Protein phosphorylation; N-glycosylation; Proteomics

The utility of ETD mass spectrometry in proteomic analysis by Leann M. Mikesh; Beatrix Ueberheide; An Chi; Joshua J. Coon; John E.P. Syka; Jeffrey Shabanowitz; Donald F. Hunt (pp. 1811-1822).

Mass spectrometry has played an integral role in the identification of proteins and their post-translational modifications (PTM). However, analysis of some PTMs, such as phosphorylation, sulfonation, and glycosylation, is difficult with collision-activated dissociation (CAD) since the modification is labile and preferentially lost over peptide backbone fragmentation, resulting in little to no peptide sequence information. The presence of multiple basic residues also makes peptides exceptionally difficult to sequence by conventional CAD mass spectrometry. Here we review the utility of electron transfer dissociation (ETD) mass spectrometry for sequence analysis of post-translationally modified and/or highly basic peptides. Phosphorylated, sulfonated, glycosylated, nitrosylated, disulfide bonded, methylated, acetylated, and highly basic peptides have been analyzed by CAD and ETD mass spectrometry. CAD fragmentation typically produced spectra showing limited peptide backbone fragmentation. However, when these peptides were fragmented using ETD, peptide backbone fragmentation produced a complete or almost complete series of ions and thus extensive peptide sequence information. In addition, labile PTMs remained intact. These examples illustrate the utility of ETD as an advantageous tool in proteomic research by readily identifying peptides resistant to analysis by CAD. A further benefit is the ability to analyze larger, non-tryptic peptides, allowing for the detection of multiple PTMs within the context of one another.

Keywords: Abbreviations; acK; acetylated lysine; AD; Alzheimer Disease; CAD; collision activated dissociation; diK; dimethylated lysine; ECD; electron capture dissociation; ESI; electrospray ionization; ETD; electron transfer dissociation; FT-ICR-MS; Fourier transform ion cyclotron resonance mass spectrometer; GlcNAc; N-acetylglucosamine; IMAC; immobilized metal affinity chromatography; LT; linear ion trap mass spectrometer; MS/MS; tandem mass spectrometry; nC; nitrosylated cysteine; PTM; post translational modification; PTR; proton transfer charge reduction; sS; sulfonated serine; pS; phosphorylated serine; gT-O; GlcNAc modified threonine; TFA; trifluoroacetic acid; triK; trimethylated lysineElectron transfer dissociation (ETD) mass spectrometry; Labile post translational modification (PTM); Phosphorylation; Sulfonation; Glycosylation; Nitrosylation; Disulfide bond; Proteomics

Proteomic analysis of phosphorylation, oxidation and nitrosylation in signal transduction by Corinne M. Spickett; Andrew R. Pitt; Nicholas Morrice; Walter Kolch (pp. 1823-1841).

Signal transduction pathways control cell fate, survival and function. They are organized as intricate biochemical networks which enable biochemical protein activities, crosstalk and subcellular localization to be integrated and tuned to produce highly specific biological responses in a robust and reproducible manner. Post translational Modifications (PTMs) play major roles in regulating these processes through a wide variety of mechanisms that include changes in protein activities, interactions, and subcellular localizations. Determining and analyzing PTMs poses enormous challenges. Recent progress in mass spectrometry (MS) based proteomics have enhanced our capability to map and identify many PTMs. Here we review the current state of proteomic PTM analysis relevant for signal transduction research, focusing on two areas: phosphorylation, which is well established as a widespread key regulator of signal transduction; and oxidative modifications, which from being primarily viewed as protein damage now start to emerge as important regulatory mechanisms.

Keywords: Abbreviations; ASK-1; apoptosis signal-regulating kinase-1; ATP; adenosine triphosphate; CAM; calmodulin; CID; collision induced dissociation; FT; Fourier transform; FT-ICR; Fourier transform-ion cyclotron resonance; Grx; glutaredoxin; GSH; glutathione; GST; glutathione S-transferase; HNE; 4-hydroxy-; trans; -2-nonenal; LC; liquid chromatography; MKP; MAPK phosphatase; MS; mass spectrometry; IMAC; immobilized metal chelate affinity chromatography; JNK; Jun N-terminal Kinase; MMTS; Methylmethanethiosulfonate; NBD-C; 7-chloro-4-nitrobenz-2-oxa-1,3-diazolel; NMDA; N; -methyl-; d; -aspartate; PTM; post-translational modification; PTP; protein tyrosine phosphatases; ROS; reactive oxygen species; SERCA; sarco/endoplasmic reticulum calcium ATPase; SHP; Src homology phosphatase; SNO; S-nitrosylated; SRM; Selected Reaction Monitoring; TOF; time of flight; Trx; thioredoxinSignal transduction; Proteomics; Phosphorylation; Oxidation; Nitrosylation

Application of different fragmentation techniques for the analysis of phosphopeptides using a hybrid linear ion trap-FTICR mass spectrometer by Christoph Stingl; Christian Ihling; Gustav Ammerer; Andrea Sinz; Karl Mechtler (pp. 1842-1852).

Electron capture dissociation (ECD) and infrared multiphoton dissociation (IRMPD) present complementary techniques for the fragmentation of peptides and proteins in Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) in addition to the commonly used collisionally activated dissociation (CAD). Both IRMPD and ECD have been shown to be applicable for an efficient sequencing of peptides and proteins, whereas ECD has proven especially valuable for mapping labile posttranslational modifications (PTMs), such as phosphorylations. In this work, we compare the different fragmentation techniques and MS detection in a linear ion trap and the ICR cell with respect to their abilities to efficiently identify and characterize phosphorylated peptides. For optimizing fragmentation parameters, sets of synthetic peptides with molecular weights ranging from approximately 1 to 4 kDa and different levels of phosphorylation were analyzed. The influence of spectrum averaging for obtaining high-quality spectra was investigated. Our results show that the fragmentation methods CAD and ECD allow for a facilitated analysis of phosphopeptides; however, their general applicability for analyzing phosphopeptides has to be evaluated in each specific case with respect to the given analytical task. The major advantage of complementary peptide cleavages by combining different fragmentation methods is the increased amount of information that is obtained during MS/MS analysis of modified peptides. On the basis of the obtained results, we are planning to design LC time-scale compatible, data-dependent MS/MS methods using the different fragmentation techniques in order to improve the identification and characterization of phosphopeptides.

Keywords: Abbreviations; AGC; automatic gain control; CAD; collisionally activated dissociation; ECD; electron capture dissociation; FT; Fourier transform; FTMS; Fourier transform mass spectrometry; ICR; ion cyclotron resonance; FTICRMS; Fourier transform ion cyclotron resonance mass spectrometry; IMAC; immobilized metal-ion affinity chromatography; IRMPD; infrared multiphoton dissociation; IT; ion trap; LC; liquid chromatography; MGF; mascot generic file; MS; Mass spectrometry; MS/MS; tandem MS; MS; ⁿ; MS to the nth power; PTM; posttranslational modification; RP-HPLC; reversed phase high-performance liquid chromatographyCAD; ECD; IRMPD; FTICR mass spectrometry; LC/MS; Phosphopeptide

Strategies for analysis of glycoprotein glycosylation by Hildegard Geyer; Rudolf Geyer (pp. 1853-1869).

Glycoproteins are known to exhibit multiple biological functions. In order to assign distinct functional properties to defined structural features, detailed information on the respective carbohydrate moieties is required. Chemical and biochemical analyses, however, are often impeded by the small amounts of sample available and the vast structural heterogeneity of these glycans, thus necessitating highly sensitive and efficient methods for detection, separation and structural investigation. The aim of this article is to briefly review suitable strategies for characterization of glycosylation at the levels of intact proteins, glycopeptides and free oligosaccharides. Furthermore, methods commonly used for isolation, fractionation and carbohydrate structure analysis of liberated glycoprotein glycans are discussed in the context of potential applications in glycoproteomics.

Keywords: Glycoproteomics; Glycosylation analysis; Lectin; Mass spectrometry; Oligosaccharide profiling; Review

Global methods for protein glycosylation analysis by mass spectrometry by Bogdan A. Budnik; Richard S. Lee; Judith A.J. Steen (pp. 1870-1880).

Mass spectrometry has been an analytical tool of choice for glycosylation analysis of individual proteins. Over the last 5 years several previously and newly developed mass spectrometry methods have been extended to global glycoprotein studies. In this review we discuss the importance of these global studies and the advances that have been made in enrichment analyses and fragmentation methods. We also briefly describe relevant sample preparation methods that have been used for the analysis of a single glycoprotein that could be extrapolated to global studies. Finally this review covers aspects of improvements and advances on the instrument front which are important to future global glycoproteomic studies.

Keywords: Global glycosylation analysis; Enrichment analysis; Chemical derivatization; Mass spectrometric methods of analysis; Precursor ion scanning; Electron capture dissociation; Electron transfer dissociation

From glycomics to functional glycomics of sugar chains: Identification of target proteins with functional changes using gene targeting mice and knock down cells of FUT8 as examples by Akihiro Kondo; Wenzhe Li; Takatoshi Nakagawa; Miyako Nakano; Nobuto Koyama; Xiangchun Wang; Jianguo Gu; Eiji Miyoshi; Naoyuki Taniguchi (pp. 1881-1889).

Comprehensive analyses of proteins from cells and tissues are the most effective means of elucidating the expression patterns of individual disease-related proteins. On the other hand, the simultaneous separation and characterization of proteins by 1-DE or 2-DE followed by MS analysis are one of the fundamental approaches to proteomic analysis. However, these analyses do not permit the complete structural identification of glycans in glycoproteins or their structural characterization. Over half of all known proteins are glycosylated and glycan analyses of glycoproteins are requisite for fundamental proteomics studies. The analysis of glycan structural alterations in glycoproteins is becoming increasingly important in terms of biomarkers, quality control of glycoprotein drugs, and the development of new drugs. However, usual approach such as proteoglycomics, glycoproteomics and glycomics which characterizes and/or identifies sugar chains, provides some structural information, but it does not provide any information of functionality of sugar chains. Therefore, in order to elucidate the function of glycans, functional glycomics which identifies the target glycoproteins and characterizes functional roles of sugar chains represents a promising approach. In this review, we show examples of functional glycomics technique using α 1,6 fucosyltransferase gene ( Fut8) in order to identify the target glycoprotein(s). This approach is based on glycan profiling by CE/MS and LC/MS followed by proteomic approaches, including 2-DE/1-DE and lectin blot techniques and identification of functional changes of sugar chains.

Keywords: Core fucosylation; Functional glycomics; Knock out; Knock down; RNAi; Lectin; MS

Protein arginine methylation: Cellular functions and methods of analysis by Steffen Pahlich; Rouzanna P. Zakaryan; Heinz Gehring (pp. 1890-1903).

During the last few years, new members of the growing family of protein arginine methyltransferases (PRMTs) have been identified and the role of arginine methylation in manifold cellular processes like signaling, RNA processing, transcription, and subcellular transport has been extensively investigated. In this review, we describe recent methods and findings that have yielded new insights into the cellular functions of arginine-methylated proteins, and we evaluate the currently used procedures for the detection and analysis of arginine methylation.

Keywords: Arginine methylation; PRMT; RNA-binding proteins; In vitro and in vivo methylation; Mass spectrometry

Protein sulfation analysis—A primer by Flavio Monigatti; Brian Hekking; Hanno Steen (pp. 1904-1913).

The aim of this review is to present an overview of protein sulfation in the context of ‘modificomics’, i.e. post-translational modification-specific proteome research. In addition to a short introduction to the biology of protein sulfation (part 1), we will provide detailed discussion regarding (i) methods and tools for prediction of protein tyrosine sulfation sites (part 2), (ii) biochemical techniques used for protein sulfation analysis (part 3.1), and (iii) mass spectrometric strategies and methods applied to protein sulfation analysis (part 3.2). We will highlight strengths and limitations of different strategies and approaches (including references), providing a primer for newcomers to protein sulfation analysis.

Keywords: Protein sulfation; Tyrosine sulfation; Mass spectrometry; Secreted protein; Trans-Golgi network; Electron capture dissociation; Electron detachment dissociation; Precursor ion scanning

Hydrophobic modifications of Ras proteins by isoprenoid groups and fatty acids—More than just membrane anchoring by Markos Pechlivanis; Juergen Kuhlmann (pp. 1914-1931).

During the last years, post-translational modification of peripheral membrane proteins with hydrophobic side groups has been attributed to a couple of additional functions than just simple anchoring into lipid bilayers. In particular isoprenylation and N- and S-acylation did quicken interest in terms of specific recognition elements for protein–protein interactions and as hydrophobic switches that allow for temporal regulated association with distinct target structures. Furthermore new insights into the heterogeneity of natural membranes have connected the physical properties of e.g. farnesyl or palmitoyl side chains with a preference for such sub-compartments as lipid rafts or caveolae. In this review the impact of the two frequently realized modifications by isoprenylation and S-acylation on the process of cellular signal transduction is exemplified with proteins of the Ras and Rab family of small GTP-binding proteins.

Keywords: Ras; Isoprenylation; Palmitoylation; Isoprenoid sensitive; Raft; Semisynthetic lipoprotein

The analysis of histone modifications by Ana Villar-Garea; Axel Imhof (pp. 1932-1939).

The biological function of many proteins is often regulated through posttranslational modifications (PTMs). Frequently different modifications influence each other and lead to an intricate network of interdependent modification patterns that affect protein–protein interactions, enzymatic activities and sub-cellular localizations. One of the best-studied class of proteins that is affected by PTMs and combinations thereof are the histone molecules. Histones are very abundant, small basic proteins that package DNA in the eukaryotic nucleus to form chromatin. The four core-histones are densely modified within their first 20–40 N-terminal amino acids, which are highly evolutionary conserved despite playing no structural role. The modifications are thought to constitute a histone code that is used by the cell to encrypt various chromatin conformations and gene expression states. The analysis of modified histones can be used as a model to dissect complex modification patterns and to investigate their molecular functions. Here we review techniques that have been used to decipher complex histone modification patterns and discuss the implication of these findings for chromatin structure and function.

Keywords: Chromatin; Histone; Mass spectrometry; Posttranslational; Modification; Lysine methylation

Dissecting the ubiquitin pathway by mass spectrometry by Ping Xu; Junmin Peng (pp. 1940-1947).

Protein modification by ubiquitin is a central regulatory mechanism in eukaryotic cells. Recent proteomics developments in mass spectrometry enable systematic analysis of cellular components in the ubiquitin pathway. Here, we review the advances in analyzing ubiquitinated substrates, determining modified lysine residues, quantifying polyubiquitin chain topologies, as well as profiling deubiquitinating enzymes based on the activity. Moreover, proteomic approaches have been developed for probing the interactome of proteasome and for identifying proteins with ubiquitin-binding domains. Similar strategies have been applied on the studies of the modification by ubiquitin-like proteins as well. These strategies are discussed with respect to their advantages, limitations and potential improvements. While the utilization of current methodologies has rapidly expanded the scope of protein modification by the ubiquitin family, a more active role is anticipated in the functional studies with the emergence of quantitative mass spectrometry.

Keywords: Abbreviations; MS; mass spectrometry; Ub; ubiquitin; AQUA; absolute quantification; LC-MS/MS; liquid chromatography coupled with tandem mass spectrometry; SRM; selective reaction monitoring; MRM; multiple reaction monitoringMass spectrometry ubiquitin

Study of posttranslational modifications in lenticular αA-Crystallin of mice using proteomic analysis techniques by Heike Schaefer; Daniel C. Chamrad; Marion Herrmann; Janine Stuwe; Gabriele Becker; Joachim Klose; Martin Blueggel; Helmut E. Meyer; Katrin Marcus (pp. 1948-1962).

In the present work the complexity in the 2D-gel protein pattern of murin lenticular αA-Crystallin was analyzed. An in depth study of the different protein isoforms was done combining different proteomic tools. Lens proteins of four different ages, from embryo to 100-week-old mice, were separated by large 2D-PAGE, revealing an increase in the number and intensity of the spots of αA-Crystallin during the process of aging. For further analyses the oldest mice were chosen. Comparison and evaluation of two different staining methods proved Imidazole-Zinc to be a good alternative to the generally used Coomassie stain. The characterization of the different αA-Crystallin protein species was done using nanoLC-ESI-MS/MS (liquid chromatography electrospray ionisation tandem mass spectrometry). Data interpretation was done by database searching, manual validation and a new MS/MS-interpretation tool for posttranslational modifications—the PTM-Explorer. Using this way, eight different phosphorylation sites were identified and localized; the identification of four of them was not published so far. Furthermore, quantitative N-terminal acetylation of αA-Crystallin and variable C-terminal truncation was observed, also not published in this extent yet. The results of the mass spectrometric analysis were validated by immunoblotting experiments using two different αA-Crystallin specific antibodies. In addition, a fluorescent phospho-specific stain was used to detect the protein spots including phosphorylation groups. Re-separation 2D-PAGE was done to round off the present study and explain the appearance of some of the protein spots in the gel as artifacts of the 2D-PAGE separation.

Keywords: αA-Crystallin; 2D-PAGE; Mass spectrometry; Phosphorylation; Posttranslational modification

Phosphoproteomics of human platelets: A quest for novel activation pathways by René P. Zahedi; Antonija J. Begonja; Stepan Gambaryan; Albert Sickmann (pp. 1963-1976).

Besides their role in hemostasis, platelets are also highly involved in the pathogenesis and progression of cardiovascular diseases. Since important and initial steps of platelet activation and aggregation are regulated by phosphorylation events, a comprehensive study aimed at the characterization of phosphorylation-driven signaling cascades might lead to the identification of new target proteins for clinical research. However, it becomes increasingly evident that only a comprehensive phosphoproteomic approach may help to characterize functional protein networks and their dynamic alteration during physiological and pathophysiological processes in platelets. In this review, we discuss current methodologies in phosphoproteome research including their potentials as well as limitations, from sample preparation to classical approaches like radiolabeling and state-of-the-art mass spectrometry techniques.

Keywords: Phosphorylation; Mass spectrometry; Proteomics; Platelet; Signal transduction

Proteins and Proteomics Cumulative Contents (pp. 1977-1987).

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BBA - Proteins and Proteomics (v.1764, #12)