Identification Of Post-translational Modifications Research Articles

Aspartic Acid Isomerization Characterized by High Definition Mass Spectrometry Significantly Alters the Bioactivity of a Novel Toxin from Poecilotheria.

Research in toxinology has created a pharmacological paradox. With an estimated 220,000 venomous animals worldwide, the study of peptidyl toxins provides a vast number of effector molecules. However, due to the complexity of the protein-protein interactions, there are fewer than ten venom-derived molecules on the market. Structural characterization and identification of post-translational modifications are essential to develop biological lead structures into pharmaceuticals. Utilizing advancements in mass spectrometry, we have created a high definition approach that fuses conventional high-resolution MS-MS with ion mobility spectrometry (HDMSE) to elucidate these primary structure characteristics. We investigated venom from ten species of “tiger” spider (Genus: Poecilotheria) and discovered they contain isobaric conformers originating from non-enzymatic Asp isomerization. One conformer pair conserved in five of ten species examined, denominated PcaTX-1a and PcaTX-1b, was found to be a 36-residue peptide with a cysteine knot, an amidated C-terminus, and isoAsp33Asp substitution. Although the isomerization of Asp has been implicated in many pathologies, this is the first characterization of Asp isomerization in a toxin and demonstrates the isomerized product’s diminished physiological effects. This study establishes the value of a HDMSE approach to toxin screening and characterization.

Middle-Down Proteomics Reveals Dense Sites of Methylation and Phosphorylation in Arginine-Rich RNA-Binding Proteins.

Post-translational modifications (PTMs) within arginine (Arg)-rich RNA-binding proteins, such as phosphorylation and methylation, regulate multiple steps in RNA metabolism. However, the identification of PTMs within Arg-rich domains with complete trypsin digestion is extremely challenging due to the high density of Arg residues within these proteins. Here, we report a middle-down proteomic approach coupled with electron-transfer dissociation (ETD) mass spectrometry to map previously unknown sites of phosphorylation and methylation within the Arg-rich domains of U1-70K and structurally similar RNA-binding proteins from nuclear extracts of human embryonic kidney (HEK)-293T cells. Notably, the Arg-rich domains in RNA-binding proteins are densely modified by methylation and phosphorylation compared with the remainder of the proteome, with methylation and phosphorylation favoring RSRS motifs. Although they favor a common motif, analysis of combinatorial PTMs within RSRS motifs indicates that phosphorylation and methylation do not often co-occur, suggesting that they may functionally oppose one another. Furthermore, we show that phosphorylation may modify interactions between Arg-rich proteins, as serine-arginine splicing factor 2 (SRSF2) has a stronger association with U1-70K and LUC7L3 upon dephosphorylation. Collectively, these findings suggest that the level of PTMs within Arg-rich domains may be among the highest in the proteome and a possible unexplored regulator of RNA-binding protein interactions.

Combination of intact, middle-up and bottom-up levels to characterize 7 therapeutic monoclonal antibodies by capillary electrophoresis – Mass spectrometry

Significant growth of biopharmaceuticals requires powerful analytical methods to better understand their structure by establishing a complete characterization. To this end, a combination of bottom-up, middle-up and intact molecule levels with a capillary electrophoresis-mass spectrometry coupling has been performed to have a comprehensive picture of monoclonal antibodies. In this study, 7 worldwide health authorities approved mAbs have been analyzed to get information about their charge heterogeneity, the identification of post translational modifications (PTMs), their location and relative quantitation. Intact mAbs isoforms have been partially separated in less than 12 min and enabled to have a global illustration of mAbs heterogeneity and high masses PTMs characterization notably major N-glycosylation forms. Particularly, 2X-glycosylated and 1X-glycosylated forms have been partially separated. To deepen characterize PTMs carried by the backbone structure, advanced investigations at a middle-up level have been performed. Limited IdeS proteolysis allowed to study independently Fc/2 and F(ab)’2 fragments. Following the same separation conditions, isoforms of these fragments have been separated and data interpretation allowed to disclose additional PTMs as K-clip, oxidations or deamidations. A second intermediate level has been examined by adding a reduction step to establish a more precise assessment of PTMs and isoforms from the F(ab)’2 fragment. This reduction step released the light chains from the Fd fragment to get only 25 kDa fragments to analyze. CE-ESI-MS coupling allowed to get more information particularly about low masses PTMs. The precise location and relative quantitation of each PTM has been investigated at the peptidic level induced by a tryptic digestion of the studied mAbs. The concordance of the results shows the efficiency of the CE-ESI-MS coupling to characterize mAbs and highlight the need of the multi-level combination to get a comprehensive characterization of biotherapeutics.

Lys-Sequencer: An algorithm for de novo sequencing of peptides by paired single residue transposed Lys-C and Lys-N digestion coupled with high-resolution mass spectrometry.

Database-dependent identification of proteins by mass spectrometry is well established, but has limitations when there are novel proteins, mutations, splice variants, and post-translational modifications (PTMs) not available in the established reference database. De novo sequencing as a database-independent approach could address these limitations by deducing peptide sequences directly from experimental tandem mass spectrometry spectra, while concomitantly yielding residue-by-residue confidence metrics. Equal amounts of bovine serum albumin (BSA) sample aliquots were digested separately with Lys-C and Lys-N complementary peptidases, separated by reversed-phase ultra-high-performance liquid chromatography (UPLC), and analyzed by collision-induced dissociation (CID)-based mass spectrometry on an Orbitrap mass spectrometer. In the Lys-Sequencer algorithm, matched tandem mass spectra with equal precursor ion mass from complementary digestions were paired, and fragment ion types were identified based on the unique mass relationship between fragment ions extracted from a spectrum pair followed by de novo sequencing of peptides with identification confidence assigned at the residue level. In all the matched spectrum pairs, 34 top-ranked BSA peptides were identified, from which 391 amino acid residues were identified correctly, covering ~67% of the full sequence of BSA (583 residues) with only ~6% (35 residues) exhibiting ambiguity in the sequence order (although amino acid compositions were still correctly assigned). Of note, this approach identified peptide sequences up to 17 amino acids in length without ambiguity, with the exception of the N-terminal or C-terminal peptides containing lysine (18-mer). The algorithm ("Lys-Sequencer") developed in this work achieves high precision for de novo sequencing of peptides. This method facilitates the identification of point mutation and new PTMs in the protein characterization and discovery of new peptides and proteins with varying levels of confidence.

Label-Free Detection of Post-translational Modifications with a Nanopore.

Post-translational modifications (PTMs) of proteins play key roles in cellular processes. Hence, PTM identification is crucial for elucidating the mechanism of complex cellular processes and disease. Here we present a method for PTM detection at the single-molecule level using FraC biological nanopores. We focus on two major PTMs, phosphorylation and glycosylation, that mutually compete for protein modification sites, an important regulatory process that has been implicated in the pathogenic pathways of many diseases. We show that phosphorylated and glycosylated peptides can be clearly differentiated from nonmodified peptides by differences in the relative current blockade and dwell time in nanopore translocations. Furthermore, we show that these PTM modifications can be mutually differentiated, demonstrating the identification of phosphorylation and glycosylation in a label-free manner. The results represent an important step for the single-molecule, label-free identification of proteoforms, which have tremendous potential for disease diagnosis and cell biology.

A mass spectrometry-based assay using metabolic labeling to rapidly monitor chromatin accessibility of modified histone proteins

Histone post-translational modifications (PTMs) contribute to chromatin accessibility due to their chemical properties and their ability to recruit enzymes responsible for DNA readout and chromatin remodeling. To date, more than 400 different histone PTMs and thousands of combinations of PTMs have been identified, the vast majority with still unknown biological function. Identification and quantification of histone PTMs has become routine in mass spectrometry (MS) but, since raising antibodies for each PTM in a study can be prohibitive, lots of potential is lost from MS datasets when uncharacterized PTMs are found to be significantly regulated. We developed an assay that uses metabolic labeling and MS to associate chromatin accessibility with histone PTMs and their combinations. The labeling is achieved by spiking in the cell media a 5x concentration of stable isotope labeled arginine and allow cells to grow for at least one cell cycle. We quantified the labeling incorporation of about 200 histone peptides with a proteomics workflow, and we confirmed that peptides carrying PTMs with extensively characterized roles in active transcription or gene silencing were in highly or poorly labeled forms, respectively. Data were further validated using next-generation sequencing to assess the transcription rate of chromatin regions modified with five selected PTMs. Furthermore, we quantified the labeling rate of peptides carrying co-existing PTMs, proving that this method is suitable for combinatorial PTMs. We focus on the abundant bivalent mark H3K27me3K36me2, showing that H3K27me3 dominantly represses histone swapping rate even in the presence of the more permissive PTM H3K36me2. Together, we envision this method will help to generate hypotheses regarding histone PTM functions and, potentially, elucidate the role of combinatorial histone codes.

Abstract 5077: Proteomic profiling reveals chemopreventive targets in esophageal adenocarcinoma

Abstract Esophageal adenocarcinoma (EAC) is characterized by rising incidence rates and high mortality due to late stage diagnosis and a lack of efficacious options for prevention and treatment. While reasons for the rapid increase in EAC are being unraveled, persistent, symptomatic reflux of gastric and duodenal contents, known as gastroesophageal reflux disease is considered the strongest risk factor. Our laboratory utilizes a rat surgical model of reflux-induced EAC to evaluate mechanisms by which cranberry proanthocyanidins (C-PAC) delivered in the drinking water inhibit reflux-induced EAC. Findings show that C-PAC (690ug/rat/day) inhibits EAC formation by 83% at 40 weeks of study and mechanisms of inhibition are under investigation. We utilized nano LC-MS/MS proteomic profiling to identify proteins that were dysregulated due to reflux-induced EAC and reversed with C-PAC treatment. Proteomic profiling revealed that C-PAC treatment restored 63 proteins dysregulated in the context of reflux. EIF3F, PSMD2 and ACTR2 were the top proteins up-regulated by reflux; whereas, top markers restored by C-PAC included GSTT2, OGN and PCMT1. Metacore integrated software showed top pathways up-regulated by reflux and down-regulated C-PAC included Translation_regulation of translation initiation (EIF-linked), Immune response and Regulation of telomere length. Conversely, C-PAC treatment upregulated the glutathione metabolism pathway which is consistent with GSTT2 restoration. Disease enrichment analysis revealed multiple gastrointestinal tract diseases linked to reflux, which C-PAC down regulated supporting that proteomic profiling may inform other potential disease targets for C-PAC. Finally, beyond the 63 reflux-driven proteins C-PAC reversed, we considered proteins, pathways and processes which could not be reversed by C-PAC (n=269). As an example, Process networks upregulated by reflux (not reversed by C-PAC) included Translation initiation (RPL-linked), Elongation, mRNA processing, G2-M and S-phase of the cell cycle. Thus, proteomics profiling may inform the development of complementary preventive combinations for improved cancer inhibitory efficacy, especially for a cancer like EAC, with high mutational burden. Western blot analysis of several markers in the rat esophagus including GSTT2 and COX2 support the data obtained through proteomic profiling. Future directions include interrogating specific pathways that are highly modulated by reflux-induced EAC and restored by C-PAC including DNA damage, repair and extracellular matrix and adhesion. Last, proteomic profiling has also allowed for identification of post-translational modifications which may provide insight into regulation of key dysregulated proteins. Citation Format: Katherine M. Weh, Connor L. Howard, Amy B. Howell, Jennifer L. Clarke, Laura A. Kresty. Proteomic profiling reveals chemopreventive targets in esophageal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5077.

One minute analysis of 200 histone posttranslational modifications by direct injection mass spectrometry.

DNA and histone proteins define the structure and composition of chromatin. Histone posttranslational modifications (PTMs) are covalent chemical groups capable of modeling chromatin accessibility, mostly due to their ability in recruiting enzymes responsible for DNA readout and remodeling. Mass spectrometry (MS)-based proteomics is the methodology of choice for large-scale identification and quantification of protein PTMs, including histones. High sensitivity proteomics requires online MS coupling with relatively low throughput and poorly robust nano-liquid chromatography (nanoLC) and, for histone proteins, a 2-d sample preparation that includes histone purification, derivatization, and digestion. We present a new protocol that achieves quantitative data on about 200 histone PTMs from tissue or cell lines in 7 h from start to finish. This protocol includes 4 h of histone extraction, 3 h of derivatization and digestion, and only 1 min of MS analysis via direct injection (DI-MS). We demonstrate that this sample preparation can be parallelized for 384 samples by using multichannel pipettes and 96-well plates. We also engineered the sequence of a synthetic “histone-like” peptide to spike into the sample, of which derivatization and digestion benchmarks the quality of the sample preparation. We ensure that DI-MS does not introduce biases in histone peptide ionization as compared to nanoLC-MS/MS by producing and analyzing a library of synthetically modified histone peptides mixed in equal molarity. Finally, we introduce EpiProfileLite for comprehensive analysis of this new data type. Altogether, our workflow is suitable for high-throughput screening of >1000 samples per day using a single mass spectrometer.

The Plant PTM Viewer, a central resource for exploring plant protein modifications.

Post-translational modifications (PTMs) of proteins are central in any kind of cellular signaling. Modern mass spectrometry technologies enable comprehensive identification and quantification of various PTMs. Given the increased numbers and types of mapped protein modifications, a database is necessary that simultaneously integrates and compares site-specific information for different PTMs, especially in plants for which the available PTM data are poorly catalogued. Here, we present the Plant PTM Viewer (http://www.psb.ugent.be/PlantPTMViewer), an integrative PTM resource that comprises approximately 370000 PTM sites for 19 types of protein modifications in plant proteins from five different species. The Plant PTM Viewer provides the user with a protein sequence overview in which the experimentally evidenced PTMs are highlighted together with an estimate of the confidence by which the modified peptides and, if possible, the actual modification sites were identified and with functional protein domains or active site residues. The PTM sequence search tool can query PTM combinations in specific protein sequences, whereas the PTM BLAST tool searches for modified protein sequences to detect conserved PTMs in homologous sequences. Taken together, these tools help to assume the role and potential interplay of PTMs in specific proteins or within a broader systems biology context. The Plant PTM Viewer is an open repository thatallows the submission of mass spectrometry-based PTM data to remain at pace with future PTM plant studies.

Advances of Proteomics in Novel PTM Discovery: Applications in Cancer Therapy

AbstractProtein posttranslational modification (PTM) is a critical mechanism to enhance the diversity of protein species and functions in organisms. Currently, many different PTMs are discovered and found to be involved in a wide range of cellular processes. The aberrant regulation of PTM dynamics is reported to contribute to cancer development. Phosphorylation and acetylation are the best studied PTMs, and the application of high‐resolution mass spectrometry‐based proteomics and specific antibody‐based enrichment technologies significantly promotes novel PTM discovery. This review discusses methods for global PTM identification, including antibody‐based enrichment and chemical probe‐based identification. Several acylations, such as propionylation, butyrylation, succinylation, malonylation, and crotonylation, are recently found to influence both histone and nonhistone proteins and to be intimately linked with the progression of cancer. It is proposed that targeting novel PTM dynamics by pharmacological inhibitors has great potential for cancer therapy.

Analysis of Methylation, Acetylation, and Other Modifications in Bacterial Ribosomal Proteins.

A wide variety of posttranslational modifications of expressed proteins are known to occur in living organisms (Krishna R, Wold F. Post-translational modification of proteins. In: Meister A (ed) Advances in enzymology and related areas of molecular biology. Wiley, New York, 1993, pp 265-296). Although their presence in an organism cannot be predicted from the genome, these modifications can play critical roles in protein structure and function. The identification of posttranslational modifications is critical to our understanding of the functions of proteins involved in important biological pathways and mass spectrometry offers a fast, accurate method for observing them. A combined top-down/bottom-up approach can be used for identification and localization of posttranslational modifications of ribosomal proteins. This chapter describes procedures for analyzing Escherichia coli ribosomal proteins and their modifications by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. It also covers the analysis of gram-negative Caulobacter crescentus and gram-positive Bacillus subtilis ribosomal proteins by electrospray quadrupole time-of-flight (ESI-QTOF) mass spectrometry. Confirmation of the occurrence and localization of PTMs is obtained through mass spectrometric analysis of tryptic peptides.

Trapping, tagging and tracking: Tools for the study of proteins during early development of the sea urchin.

The exquisite synchronicity of sea urchin development provides a reliable model for studying maternal proteins in the haploid egg as well as those involved in egg activation, fertilization and early development. Sea urchin eggs are released by the millions, enabling the quantitative evaluation of maternally stored and newly synthesized proteins over a range of time (seconds to hours post fertilization). During this window of development exist many hallmark and unique biochemical interactions that can be investigated for the purpose of characterizing profiles of kinases and other signaling proteins, manipulated using pharmacology to test sufficiency and necessity, for identification of post translational modifications, and for capturing protein-protein interactions. Coupled with the fact that sea urchin eggs and embryos are transparent, this synchronicity also results in large populations of cells that can be evaluated for newly synthesized protein localization and identification through use of the Click-iT technology. We provide basic protocols for these approaches and direct readers to the appropriate literature for variations and examples.

Changing times: Fluorescence-lifetime analysis of amyloidogenic SF-IAPP fusion protein

In a number of conformational diseases, intracellular accumulation of proteins bearing non-native conformations occurs. The search for compounds that are capable of hindering the formation and accumulation of toxic protein aggregates and fibrils is an urgent task. Present fluorescent methods of fibrils’ detection prevent simple real-time observations. We suppose to use green fluorescent protein fused with target protein and fluorescence lifetime measurement technique for this purpose.The recombinant proteins analyzed were produced in E. coli. Mass spectrometry was used for the primary structure of the recombinant proteins and post-translational modifications identification. The fluorescence lifetime of the superfolder green fluorescent protein (SF) and the SF protein fused with islet amyloid polypeptide (SF-IAPP) were studied in polyacrylamide gel using Fluorescent-Lifetime Imaging Microscopy (FLIM).It was shown that the SF average fluorescence lifetime in gel slightly differs from that of the SF-IAPP monomer under these conditions. SF-IAPP does not lose the ability to form amyloid-like fibrils. Under the same conditions (in polyacrylamide gel), SF and SF-IAPP monomers have similar fluorescence time characteristics and the average fluorescence lifetime of SF-IAPP in fibrils significantly decreases.We propose the application of FLIM to the measurement of average fluorescence lifetimes of fusion proteins (amyloidogenic protein-SF) in the context of studies using cellular models of conformational diseases.

A Systematic Review on Posttranslational Modification in Proteins: Feature Construction, Algorithm and Webserver

Proteins through post translational modifications perform their biological process and cellular functions. Post-translational modifications play important roles in various biological process and cell functions. Identifying the PTMs sites in proteins is very significant to basic research and drug design. Experimental technique to identify post translational modifications is laborious. Computational identification of post translational modifications is a complementary way for its convenience. This review gives the processing to predict post-translational modification sites in proteins including feature construction, algorithms, evaluation measurement, and online webserver. There are two types of post translational modification in proteins. In the prediction of single PTM sites, we transformed it into binary classification learning. While in the prediction of crosstalk PTM sites, we transformed it into multi-label learning. This review summarized the steps on the two issues.

Extensive Characterization of Heavily Modified Histone Tails by 193 nm Ultraviolet Photodissociation Mass Spectrometry via a Middle-Down Strategy.

The ability to map combinatorial patterns of post-translational modifications (PTMs) of proteins remains challenging for traditional bottom-up mass spectrometry workflows. There are also hurdles associated with top-down approaches related to limited data analysis options for heavily modified proteoforms. These shortcomings have accelerated interest in middle-down MS methods that focus on analysis of large peptides generated by specific proteases in conjunction with validated bioinformatics strategies to allow quantification of isomeric histoforms. Mapping multiple PTMs simultaneously requires the ability to obtain high sequence coverage to allow confident localization of the modifications, and 193 nm ultraviolet photodissociation (UVPD) has been shown to cause extensive fragmentation for large peptides and proteins. Histones are an ideal system to test the ability of UVPD to characterize multiple modifications, as the combinations of PTMs are the underpinning of the biological significance of histones and at the same time create an imposing challenge for characterization. The present study focuses on applying 193 nm UVPD to the identification and localization of PTMs on histones by UVPD and comparison to a popular alternative, electron-transfer dissociation (ETD), via a high-throughput middle-down LC/MS/MS strategy. Histone Coder and IsoScale, bioinformatics tools for verification of PTM assignments and quantification of histone peptides, were adapted for UVPD data and applied in the present study. In total, over 300 modified forms were identified, and the distributions of PTMs were quantified between UVPD and ETD. Significant differences in patterns of PTMs were found for histones from HeLa cells prior to and after treatment with a deacetylase inhibitor. Additional fragment ion types generated by UVPD proved essential for extensive characterization of the most heavily modified forms (>5 PTMs).

P-74 - Uncovering the diversity of redox proteoforms and their significance in cellular signaling and protein-protein interactions

Existence of numerous proteoforms, different molecular forms of a protein coded by a single gene, derived by genetic variations, alternative splicing and post-translational modifications (PTMs), is proposed to be responsible for the functional and structural diversity of protein species in biological systems. Till now protein phosphorylation and acetylation are the most studied PTMs with respect to proteoforms variety, functional significance and possible PTM cross-talks. However, much less attention is paid to the redox-derived modifications. Using state-of-the-art mass spectrometry-based proteomics, we performed deep profiling of redox modifications in a variety of oxidative stress model systems as well as clinical samples, to uncover the possible impact of oxidative PTMs on protein structure and functions. Dynamic alterations in over 60 different modifications covering ten amino acid residues were demonstrated for a number of proteins including actin, vimentin, and cytochrome c providing first insights in redox proteoforms diversity and PTM cross-talks in biological systems. PTM identification was complemented by cheminformatics, biochemical and fluorescent microscopy studies, to correlate modification dynamics with structural and functional outcomes.

Investigation and identification of functional post-translational modification sites associated with drug binding and protein-protein interactions

BackgroundProtein post-translational modification (PTM) plays an essential role in various cellular processes that modulates the physical and chemical properties, folding, conformation, stability and activity of proteins, thereby modifying the functions of proteins. The improved throughput of mass spectrometry (MS) or MS/MS technology has not only brought about a surge in proteome-scale studies, but also contributed to a fruitful list of identified PTMs. However, with the increase in the number of identified PTMs, perhaps the more crucial question is what kind of biological mechanisms these PTMs are involved in. This is particularly important in light of the fact that most protein-based pharmaceuticals deliver their therapeutic effects through some form of PTM. Yet, our understanding is still limited with respect to the local effects and frequency of PTM sites near pharmaceutical binding sites and the interfaces of protein-protein interaction (PPI). Understanding PTM’s function is critical to our ability to manipulate the biological mechanisms of protein.ResultsIn this study, to understand the regulation of protein functions by PTMs, we mapped 25,835 PTM sites to proteins with available three-dimensional (3D) structural information in the Protein Data Bank (PDB), including 1785 modified PTM sites on the 3D structure. Based on the acquired structural PTM sites, we proposed to use five properties for the structural characterization of PTM substrate sites: the spatial composition of amino acids, residues and side-chain orientations surrounding the PTM substrate sites, as well as the secondary structure, division of acidity and alkaline residues, and solvent-accessible surface area. We further mapped the structural PTM sites to the structures of drug binding and PPI sites, identifying a total of 1917 PTM sites that may affect PPI and 3951 PTM sites associated with drug-target binding. An integrated analytical platform (CruxPTM), with a variety of methods and online molecular docking tools for exploring the structural characteristics of PTMs, is presented. In addition, all tertiary structures of PTM sites on proteins can be visualized using the JSmol program.ConclusionResolving the function of PTM sites is important for understanding the role that proteins play in biological mechanisms. Our work attempted to delineate the structural correlation between PTM sites and PPI or drug-target binding. CurxPTM could help scientists narrow the scope of their PTM research and enhance the efficiency of PTM identification in the face of big proteome data. CruxPTM is now available at http://csb.cse.yzu.edu.tw/CruxPTM/.

Identification of Post-translational Modifications on Odorant-Binding Protein Isoforms from Pig Olfactory Secretome by High-Resolution Mass Spectrometry: O-β-N-acetylglucosaminylation and Phosphorylation

Odorant-Binding Proteins (OBP) are major players of perireceptor events in olfaction. Despite their importance, a molecular mechanism explaining their specificity for odors and pheromones has yet to be proposed. A new approach is provided by the analysis of the pig olfactory secretome that is mainly composed of OBP isoforms, generated from 3 gene products by two types of post-translational modifications (PTM): i) phosphorylation and ii) O--N-acetylglucosaminylation (O-GlcNAcylation), which are unusual for secreted proteins. Although both types of PTM can be demonstrated on OBP isoforms by specific antibodies, they have to be identified by mass spectrometry (MS), as localizing PTM sites and identifying PTM patterns can help predict binding affinities. In this paper, we report the identification of phosphorylation and O-GlcNAcylation sites on peptides coming from trypsin digestion of only OBP (sensu stricto) by nanoLC-nanoESI-HCD-MS/MS-Orbitrap. These PTM were not present on VEG and SAL. PEAKS software analysis of raw MS data allowed selecting spectra that were analyzed manually to identify PTMs. Four peptides corresponding to two different portions of OBP sequence were modified either by a phosphate group or by a hexNAc moiety. Due to the high energy used in HCD, the data did not allow precise localization of the modified sites. The new findings contribute to a better understanding of the mechanisms by which OBP isoforms could extend the binding repertoire of the secreted OBPs. Data are available via ProteomeXchange with identifier PXD007955.

Histone H1 Purification and Post-Translational Modification Profiling by High-Resolution Mass Spectrometry.

It has proven particularly difficult to purify Linker (H1) histones from the model plant Arabidopsis thaliana. This is most likely due to its low nuclear DNA content and the abundance of substances that interfere with protein isolation. These problems have hindered the use of Arabidopsis for in-depth characterization of nuclear proteins by modern techniques based on mass spectrometry (MS). Here, we describe an improved methodology for preparing pure Arabidopsis H1s and separating them by HPLC into fractions corresponding to nonallelic variants. In addition, we outline basic approaches enabling the identification of posttranslational modifications of H1 by MS and their mapping by digestion with different proteases. We also discuss the analysis and interpretation of the acquired data.

Optimizing High-Resolution Mass Spectrometry for the Identification of Low-Abundance Post-Translational Modifications of Intact Proteins.

Intact protein analysis by liquid chromatography-mass spectrometry (LC-MS) is now possible due to the improved capabilities of mass spectrometers yielding greater resolution, mass accuracy, and extended mass ranges. Concurrent measurement of post-translational modifications (PTMs) during LC-MS of intact proteins is advantageous while monitoring critical proteoform status, such as for clinical samples or during production of reference materials. However, difficulties exist for PTM identification when the protein is large or contains multiple modification sites. In this work, analyses of low-abundance proteoforms of proteins of clinical or therapeutic interest, including C-reactive protein, vitamin D-binding protein, transferrin, and immunoglobulin G (NISTmAb), were performed on an Orbitrap Elite mass spectrometer. This work investigated the effect of various instrument parameters including source temperatures, in-source CID, microscan type and quantity, resolution, and automatic gain control on spectral quality. The signal-to-noise ratio was found to be a suitable spectral attribute which facilitated identification of low abundance PTMs. Source temperature and CID voltage were found to require specific optimization for each protein. This study identifies key instrumental parameters requiring optimization for improved detection of a variety of PTMs by LC-MS and establishes a methodological framework to ensure robust proteoform identifications, the first step in their ultimate quantification.