Top‐down mass spectrometry for the analysis of combinatorial post‐translational modifications

Here, we present a detailed PTM 1 characterization of a series of the main and clinically relevant allergens used in allergy tests and vaccines. We employ Orbitrap-based mass spectrometry with complementary fragmentation techniques (HCD/ETD) for site-specific PTM characterization by bottom-up analysis. In addition, top-down mass spectrometry is utilized for targeted analysis of individual proteins, revealing hitherto unknown PTMs of HDM allergens. We demonstrate the presence of lysine-linked polyhexose glycans and asparagine-linked N-acetylhexosamine glycans on HDM allergens. Moreover, we identified more complex glycan structures than previously reported on the major grass pollen group 1 and 5 allergens, implicating important roles for carbohydrates in allergen recognition and response by the immune system. The new findings are important for understanding basic diseasecausing mechanisms at the cellular level, which ultimately may pave the way for instigating novel approaches for targeted desensitization strategies and improved allergy vaccines. Molecular & Cellular

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“…S4B-4C 28 . Taken together, the HCD results show that the first and second hydroxylation occurs on P 31 and P 28 , respectively.…”

Section: Resultsmentioning

confidence: 99%

Glycoproteomic Analysis of Seven Major Allergenic Proteins Reveals Novel Post-translational Modifications

Halim

Carlsson

Madsen

et al. 2015

Molecular & Cellular Proteomics

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“…The identification, localization, and quantification of different combinations of PTMs on the same protein can sometimes be better solved by so-called top-down or middle-down proteomics, as opposed to the more common bottom-up proteomics (Lanucara and Eyers, 2013;Catherman et al, 2014). Bottom-up proteomics involves the cleavage of proteins into smaller fragments (by proteolysis or chemical cleavage) prior to MS analysis.…”

Section: Proteins With Multiple Ptms Combinatorial Ptms and Cross Tmentioning

confidence: 99%

Update: Post-translational protein modifications in plant metabolism

2015

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ORCID ID: 0000-0001-9536-0487 (K.J.v.W.).Posttranslational modifications (PTMs) of proteins greatly expand proteome diversity, increase functionality, and allow for rapid responses, all at relatively low costs for the cell. PTMs play key roles in plants through their impact on signaling, gene expression, protein stability and interactions, and enzyme kinetics. Following a brief discussion of the experimental and bioinformatics challenges of PTM identification, localization, and quantification (occupancy), a concise overview is provided of the major PTMs and their (potential) functional consequences in plants, with emphasis on plant metabolism. Classic examples that illustrate the regulation of plant metabolic enzymes and pathways by PTMs and their cross talk are summarized. Recent large-scale proteomics studies mapped many PTMs to a wide range of metabolic functions. Unraveling of the PTM code, i.e. a predictive understanding of the (combinatorial) consequences of PTMs, is needed to convert this growing wealth of data into an understanding of plant metabolic regulation.The primary amino acid sequence of proteins is defined by the translated mRNA, often followed by Nor C-terminal cleavages for preprocessing, maturation, and/or activation. Proteins can undergo further reversible or irreversible posttranslational modifications (PTMs) of specific amino acid residues. Proteins are directly responsible for the production of plant metabolites because they act as enzymes or as regulators of enzymes. Ultimately, most proteins in a plant cell can affect plant metabolism (e.g. through effects on plant gene expression, cell fate and development, structural support, transport, etc.). Many metabolic enzymes and their regulators undergo a variety of PTMs, possibly resulting in changes in oligomeric state, stabilization/degradation, and (de)activation (Huber and Hardin, 2004), and PTMs can facilitate the optimization of metabolic flux. However, the direct in vivo consequence of a PTM on a metabolic enzyme or pathway is frequently not very clear, in part because it requires measurements of input and output of the reactions, including flux through the enzyme or pathway. This Update will start out with a short overview on the major PTMs observed for each amino acid residue (Table I), followed by a brief discussion of techniques for the characterization of protein PTMs, including determination of the localization within proteins (i.e. the specific residues) and occupancy. Challenges in dealing with multiple PTMs per protein and cross talk between PTMs will be briefly outlined. We then describe the major physiological PTMs observed in plants as well as PTMs that are nonenzymatically induced during sample preparation (Table II). Finally, this Update is completed with a number of well-studied, classical examples of the regulation of plant metabolism by PTMs, in particular for enzymes in primary metabolism (Calvin cycle, glycolysis, and respiration) and the C4 shuttle accommodating photosynthesis in C4 plants (Table III). As will be ev...

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“…Intact protein sequencing [107] was difficult due to its large molecular weight, and requires more expensive MS instrumentation, which hinders the implementation of this technique in a typical academic laboratory. However nowadays, some top-down approaches are used in proteomics such as analyzing PTMs [65], mapping intact protein isoforms [124], etc. Some researchers also combined bottom-up and top-down mass spectrometry analysis of the histone PTMs, and their combinatorial patterns [18].…”

Section: Research Objective and Motivationmentioning

confidence: 99%

NBPMF: Novel network-based inference methods for peptide mass fingerprinting

Liang

Lajoie

Zhang

2017

2017 IEEE 16th International Conference on Cognitive Informatics &Amp; Cognitive Computing (ICCI*CC)

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Proteins are large, complex molecules that perform a vast array of functions in every living cell. A proteome is a set of proteins produced in an organism, and proteomics is the large-scale study of proteomes. Several high-throughput technologies have been developed in proteomics, where the most commonly applied are mass spectrometry (MS) based approaches.MS is an analytical technique for determining the composition of a sample. Recently it has become a primary tool for protein identification, quantification, and post translational modification (PTM) characterization in proteomics research. There are usually two different ways to identify proteins: top-down and bottom-up. Top-down approaches are based on subjecting intact protein ions and large fragment ions to tandem MS directly, while bottom-up methods are based on mass spectrometric analysis of peptides derived from proteolytic digestion, usually with trypsin.In bottom-up techniques, peptide mass fingerprinting (PMF) is widely used to identify proteins from MS dataset. Conventional PMF representatives such as probabilistic MOWSE algorithm, is based on mass distribution of tryptic peptides. In this thesis, we developed a novel network-based inference software termed NBPMF. By analyzing peptide-protein bipartite network, we designed new peptide protein matching score functions. We present two methods: the static one, ProbS, is based on an independent probability framework; and the dynamic one, HeatS, depicts input dataset as dependent peptides. Moreover, we use linear regression to adjust the matching score according to the masses of proteins. In addition, we consider the order of retention time to further correct the score function. In the post processing, we design two algorithms: assignment of peaks, and protein filtration. The former restricts that a peak can only be assigned to one peptide in order to reduce random matches; and the latter assumes each peak can only be assigned to one protein. In the result validation, we propose two new target-decoy search strategies to estimate the false discovery rate (FDR). The experiments on simulated, authentic, and simulated authentic dataset demonstrate that our NBPMF approaches lead to significantly improved performance compared to several state-of-the-art methods.i

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Top‐down mass spectrometry for the analysis of combinatorial post‐translational modifications

Cited by 60 publications

References 107 publications

Glycoproteomic Analysis of Seven Major Allergenic Proteins Reveals Novel Post-translational Modifications

Glycoproteomic Analysis of Seven Major Allergenic Proteins Reveals Novel Post-translational Modifications

Update: Post-translational protein modifications in plant metabolism

NBPMF: Novel network-based inference methods for peptide mass fingerprinting

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