Simultaneous Affinity Enrichment of Two Post-Translational Modifications for Quantification and Site Localization.

Serial and multi-level proteome analysis for microscale protein samples

Redox-active cysteine, a highly reactive sulfhydryl, is one of the major targets of ROS. Formation of disulfide bonds and other oxidative derivatives of cysteine including sulfenic, sulfinic, and sulfonic acids, regulates the biological function of various proteins. We identified novel low-abundant cysteine modifications in cellular GAPDH purified on 2-dimensional gel electrophoresis (2D-PAGE) by employing selectively excluded mass screening analysis for nano ultraperformance liquid chromatography-electrospray-quadrupole-time of flight tandem mass spectrometry, in conjunction with MODi and MODmap algorithm. We observed unexpected mass shifts (Δm=-16, -34, +64, +87, and +103 Da) at redox-active cysteine residue in cellular GAPDH purified on 2D-PAGE, in oxidized NDP kinase A, peroxiredoxin 6, and in various mitochondrial proteins. Mass differences of -16, -34, and +64 Da are presumed to reflect the conversion of cysteine to serine, dehydroalanine (DHA), and Cys-SO2-SH respectively. To determine the plausible pathways to the formation of these products, we prepared model compounds and examined the hydrolysis and hydration of thiosulfonate (Cys-S-SO2-Cys) either to DHA (Δm=-34 Da) or serine along with Cys-SO2-SH (Δm=+64 Da). We also detected acrylamide adducts of sulfenic and sulfinic acids (+87 and +103 Da). These findings suggest that oxidations take place at redox-active cysteine residues in cellular proteins, with the formation of thiosulfonate, Cys-SO2-SH, and DHA, and conversion of cysteine to serine, in addition to sulfenic, sulfinic and sulfonic acids of reactive cysteine.

Dynamic posttranslational modifications of cytoskeletal proteins unveil hot spots under nitroxidative stress

Post-translational modifications (PTMs) of proteins play crucial roles in defining protein function. They often do not occur alone, leading to a large variety of proteoforms that correspond to different combinations of multiple PTMs simultaneously decorating a protein. Changes of these proteoforms can be quantified via middle-down and top-down mass spectrometry experiments where the simultaneous PTM settings are obtained by measuring long peptides or entire proteins. Data from such experiments poses big challenges in identifying relevant features of biological and clinical importance. Generally, multiple data layers need to be considered such as proteoforms, individual PTMs, and PTM types. Therein, visualization methods are a crucial part of data analysis as they provide, if applied correctly, insights into both general behaviors as well as a deep view into fine-grained behavior. Here, we present a workflow to visualize histone proteins and their myriad of PTMs based on different R visualization modules applied to data from quantitative middle-down experiments. The procedure can be adapted to diverse experimental designs and is applicable to different proteins and PTMs.

Liquid chromatography(LC) tandem mass spectrometry (MS/MS) isone of the widely used proteomic techniques to study the alterationsoccurring at the protein level as well as post-translational modifications(PTMs) of proteins that are relevant to different physiological orpathological statuses. The mass spectrometric analysis of peptidesdigested from proteins (bottom-up proteomics) has emerged as one ofthe major approaches for proteomics. In this approach, proteins arefirst cleaved by one or more proteases into peptides for MS analysis,and peptides with PTMs are further enriched, followed by the LC-MS/MSanalysis. To achieve a reproducible and quantitative proteomic characterization,a well-established protease digestion and PTM peptide enrichment protocolis critical. In this study, we developed AUTO-SP, a sample preparationplatform providing automated protocols for BCA analysis, protein digestion,and PTM enrichment for protein and PTM analyses. We utilized patient-derivedxenograft (PDX) breast cancer tumor tissues (basal-like and luminalsubtypes) to demonstrate the efficacy of AUTO-SP. The protein amountwas quantified, and proteins were further digested by using AUTO-SPfor each PDX sample. Based on the data-independent acquisition (DIA)-MSdata, we observed that samples of the same breast cancer subtypeswere highly correlated (≥0.98). Additionally, >25,000 phosphopeptidesand >14,000 ubiquitinated peptides were identified in the PDX sampleswhen using AUTO-SP for PTM enrichment, while unique pathways wereenriched from the differentially expressed ubiquitinated peptidesof basal-like and luminal subtypes. AUTO-SP demonstrated its efficacyto provide a reliable and reproducible sample preparation procedurefor MS-based proteomic and PTM analyses.

Post-translational protein modifications can be recognized by B and T lymphocytes and can potentially make "self"-proteins appear foreign to the immune system. Such modifications may directly affect major histocompatibility complex-restricted T cell recognition of processed peptides or may perturb the processing events that generate such peptides. Using the tetanus toxin C fragment protein as a test case, we show that spontaneous deamidation of asparagine residues interferes with processing by the enzyme asparagine endopeptidase (AEP) and contributes to diminished antigen presentation. Deamidation inhibits AEP action either directly, when asparagine residues targeted by AEP are modified, or indirectly, when adjacent Asn residues are deamidated. Thus, deamidation of long-lived self-proteins may qualitatively or quantitatively affect the spectrum of self-peptides displayed to T cells and may thereby contribute to the onset or exacerbation of autoimmune disease.

Post-translational modification (PTM) of proteins regulates cellular proteostasis by expanding protein functional diversity. This naturally leads to increased proteome complexity as a result of PTM crosstalk. Here, we used the molecular chaperone protein, Heat shock protein-90 (Hsp90), which is subject to a plethora of PTMs, to investigate this concept. Hsp90 is at the hub of proteostasis and cellular signaling networks in cancer and is, therefore, an attractive therapeutic target in cancer. We demonstrated that deletion of histone deacetylase 3 (HDAC3) and histone deacetylase 8 (HDAC8) in human cells led to increased binding of Hsp90 to both ATP and its ATP-competitive inhibitor, Ganetespib. When bound to this inhibitor, Hsp90 from both HDAC3 and HDAC8 knock-out human cells exhibited similar PTMs, mainly phosphorylation and acetylation, and created a common proteomic network signature. We used both a deep-learning artificial intelligence (AI) prediction model and data based on mass spectrometry analysis of Hsp90 isolated from the mammalian cells bound to its drugs to decipher PTM crosstalk. The alignment of data from both methods demonstrates that the deep-learning prediction model offers a highly efficient and rapid approach for deciphering PTM crosstalk on complex proteins such as Hsp90.

The increasing dynamic functions of post-translational modifications (PTMs) within protein molecules present outstanding challenges for plant biology even at this present day. Protein PTMs are among the first and fastest plant responses to changes in the environment, indicating that the mechanisms and dynamics of PTMs are an essential area of plant biology. Besides being key players in signaling, PTMs play vital roles in gene expression, gene, and protein localization, protein stability and interactions, as well as enzyme kinetics. In this review, we take a broader but concise approach to capture the current state of events in the field of plant PTMs. We discuss protein modifications including citrullination, glycosylation, phosphorylation, oxidation and disulfide bridges, N-terminal, SUMOylation, and ubiquitination. Further, we outline the complexity of studying PTMs in relation to compartmentalization and function. We conclude by challenging the proteomics community to engage in holistic approaches towards identification and characterizing multiple PTMs on the same protein, their interaction, and mechanism of regulation to bring a deeper understanding of protein function and regulation in plants.

The receptor tyrosine kinase ErbB2 is a breast cancer biomarker whose posttranslational modifications (PTMs) are a key indicator of its activation. Quantifying the expression and PTMs of biomarkers such as ErbB2 by selected reaction monitoring (SRM) mass spectrometry has several limitations, including minimal coverage and extensive assay development time. Therefore, we assessed the utility of two high resolution, full scan mass spectrometry approaches, MS1 Filtering and SWATH MS2, for targeted ErbB2 proteomics. Endogenous ErbB2 immunoprecipitated from SK-BR-3 cells was in-gel digested with trypsin, chymotrypsin, Asp-N, or trypsin plus Asp-N in triplicate. Data-dependent acquisition with an AB SCIEX TripleTOF 5600 and MS1 Filtering data processing was used to assess peptide and PTM coverage as well as the reproducibility of enzyme digestion. Data-independent acquisition (SWATH) was also performed for MS2 quantitation. MS1 Filtering and SWATH MS2 allow quantitation of all detected analytes after acquisition, enabling the use of multiple proteases for quantitative assessment of target proteins. Combining high resolution proteomics with multiprotease digestion enabled quantitative mapping of ErbB2 with excellent reproducibility, improved amino acid sequence and PTM coverage, and decreased assay development time compared to typical SRM assays. These results demonstrate that high resolution quantitative proteomic approaches are an effective tool for targeted biomarker quantitation.

提出了地震微观前兆预报网络系统设计的一整套详细方案，包括地震模型、基岩传感器网络和数据融合、信号处理技术和网络系统设计。本文介绍网络系统设计方案，包括光纤传感器在地震预报网络中的应用、传感器网络平台的选择和网络设计、地震源定位的相干声阵列处理技术、光数据通信技术。

We present the first large scale study characterizing both N- and O-linked glycosylation in a site-specific manner on hundreds of proteins. We demonstrate that a lectin-affinity fractionation step using wheat germ agglutinin enriches not only peptides carrying intracellular O-GlcNAc, but also those bearing ER/Golgi-derived N- and O-linked carbohydrate structures. Liquid chromatography-MS (LC/MS) analysis with high accuracy precursor mass measurements and high sensitivity ion trap electron-transfer dissociation (ETD) were utilized for structural characterization of glycopeptides. Our results reveal both the identity of the precise sites of glycosylation and information on the oligosaccharide structures possible on these proteins. We report a novel iterative approach that allowed us to interpret the ETD data set directly without making prior assumptions about the nature and distribution of oligosaccharides present in our glycopeptide mixture. Over 2500 unique N- and O-linked glycopeptides were identified on 453 proteins. The extent of microheterogeneity varied extensively, and up to 19 different oligosaccharides were attached at a given site. We describe the presence of the well-known mucin-type structures for O-glycosylation, an EGF-domain-specific fucosylation and a rare O-mannosylation on the transmembrane phosphatase Ptprz1. Finally, we identified three examples of O-glycosylation on tyrosine residues.

Post-translational modification by small ubiquitin-like modifier 1 (SUMO-1) is a highly conserved process from yeast to humans and plays important regulatory roles in many cellular processes. Sumoylation occurs at certain internal lysine residues of target proteins via an isopeptide bond linkage. Unlike ubiquitin whose carboxyl-terminal sequence is RGG, the tripeptide at the carboxyl terminus of SUMO is TGG. The presence of the arginine residue at the carboxyl terminus of ubiquitin allows tryptic digestion of ubiquitin conjugates to yield a signature peptide containing a diglycine remnant attached to the target lysine residue and rapid identification of the ubiquitination site by mass spectrometry. The absence of lysine or arginine residues in the carboxyl terminus of mammalian SUMO makes it difficult to apply this approach to mapping sumoylation sites. We performed Arg scanning mutagenesis by systematically substituting amino acid residues surrounding the diglycine motif and found that a SUMO variant terminated with RGG can be conjugated efficiently to its target protein under normal sumoylation conditions. We developed a Programmed Data Acquisition (PDA) mass spectrometric approach to map target sumoylation sites using this SUMO variant. A web-based computational program designed for efficient identification of the modified peptides is described.

BackgroundLysine carbamylation is a biomarker of rheumatoid arthritis and kidney diseases. However, its cellular function is understudied due to the lack of tools for systematic analysis of this post-translational modification (PTM).MethodsWe adapted a method to analyze carbamylated peptides by co-affinity purification with acetylated peptides based on the cross-reactivity of anti-acetyllysine antibodies. We also performed immobilized-metal affinity chromatography to enrich for phosphopeptides, which allowed us to obtain multi-PTM information from the same samples.ResultsBy testing the pipeline with RAW 264.7 macrophages treated with bacterial lipopolysaccharide, 7,299, 8,923 and 47,637 acetylated, carbamylated, and phosphorylated peptides were identified, respectively. Our analysis showed that carbamylation occurs on proteins from a variety of functions on sites with similar as well as distinct motifs compared to acetylation. To investigate possible PTM crosstalk, we integrated the carbamylation data with acetylation and phosphorylation data, leading to the identification 1,183 proteins that were modified by all 3 PTMs. Among these proteins, 54 had all 3 PTMs regulated by lipopolysaccharide and were enriched in immune signaling pathways, and in particular, the ubiquitin-proteasome pathway. We found that carbamylation of linear diubiquitin blocks the activity of the anti-inflammatory deubiquitinase OTULIN.ConclusionsOverall, our data show that anti-acetyllysine antibodies can be used for effective enrichment of carbamylated peptides. Moreover, carbamylation may play a role in PTM crosstalk with acetylation and phosphorylation, and that it is involved in regulating ubiquitination in vitro.Graphical 9DSWmM7hoJm8b3LaxSLrsCVideo

Most naturally occurring mammalian cancers and immortalized tissue culture cell lines share a common characteristic, the overexpression of full-length HMGA1 (high mobility group A1) proteins. The HMGA1 protooncogene codes for two closely related isoform proteins, HMGA1a and HMGA1b, and causes cancerous cellular transformation when overexpressed in either transgenic mice or "normal" cultured cell lines. Previous work has suggested that the in vivo types and patterns of the HMGA1 post-translational modifications (PTMs) differ between normal and malignant cells. The present study focuses on the important question of whether HMGA1a and HMGA1b proteins isolated from the same cell type have identical or different PTM patterns and also whether these isoform patterns differ between non-malignant and malignant cells. Two independent mass spectrometry methods were used to identify the types of PTMs found on specific amino acid residues on the endogenous HMGA1a and HMGA1b proteins isolated from a non-metastatic human mammary epithelial cell line, MCF-7, and a malignant metastatic cell line derived from MCF-7 cells that overexpressed the transgenic HMGA1a protein. Although some of the PTMs were the same on both the HMGA1a and HMGA1b proteins isolated from a given cell type, many other modifications were present on one but not the other isoform. Furthermore, we demonstrate that both HMGA1 isoforms are di-methylated on arginine and lysine residues. Most importantly, however, the PTM patterns on the endogenous HMGA1a and HMGA1b proteins isolated from non-metastatic and metastatic cells were consistently different, suggesting that the isoforms likely exhibit differences in their biological functions/activities in these cell types.

Posttranslational modifications (PTMs) regulate molecular structures and functions of proteins by covalently binding to amino acids. Hundreds of thousands of PTMs have been reported for the human proteome, with multiple PTMs known to affect tens of thousands of lysine (K) residues. Our molecular evolutionary analyses show that K residues with multiple PTMs exhibit greater conservation than those with a single PTM, but the difference is rather small. In contrast, short-term evolutionary trends revealed in an analysis of human population variation exhibited a much larger difference. Lysine residues with three PTMs show 1.8-fold enrichment of Mendelian disease-associated variants when compared with K residues with two PTMs, with the latter showing 1.7-fold enrichment of these variants when compared with the K residues with one PTM. Rare polymorphisms in humans show a similar trend, which suggests much greater negative selection against mutations of K residues with multiple PTMs within population. Conversely, common polymorphisms are overabundant at unmodified K residues and at K residues with fewer PTMs. The observed difference between inter- and intraspecies patterns of purifying selection on residues with PTMs suggests extensive species-specific drifting of PTM positions. These results suggest that the functionality of a protein is likely conserved, without necessarily conserving the PTM positions over evolutionary time.