Calsequestrins in skeletal and cardiac muscle from adult Danio rerio

Calsequestrin (Casq) is a high capacity, low affinity Ca2+-binding protein, critical for Ca2+-buffering in cardiac and skeletal muscle sarcoplasmic reticulum. All vertebrates have multiple genes encoding for different Casq isoforms. Increasing interest has been focused on mammalian and human Casq genes since mutations of both cardiac (Casq2) and skeletal muscle (Casq1) isoforms cause different, and sometime severe, human pathologies. Danio rerio (zebrafish) is a powerful model for studying function and mutations of human proteins. In this work, expression, biochemical properties cellular and sub-cellular localization of D. rerio native Casq isoforms are investigated. By quantitative PCR, three mRNAs were detected in skeletal muscle and heart with different abundances. Three zebrafish Casqs: Casq1a, Casq1b and Casq2 were identified by mass spectrometry (Data are available via ProteomeXchange with identifier PXD002455). Skeletal and cardiac zebrafish calsequestrins share properties with mammalian Casq1 and Casq2. Skeletal Casqs were found primarily, but not exclusively, at the sarcomere Z-line level where terminal cisternae of sarcoplasmic reticulum are located

Accurate and Automated High-Coverage Identification of Chemically Cross-Linked Peptides with MaxLynx

Cross-linking combined with mass spectrometry (XL-MS) provides a wealth of information about the three-dimensional (3D) structure of proteins and their interactions. We introduce MaxLynx, a novel computational proteomics workflow for XL-MS integrated into the MaxQuant environment. It is applicable to noncleavable and MS-cleavable cross-linkers. For both, we have generalized the Andromeda peptide database search engine to efficiently identify cross-linked peptides. For noncleavable peptides, we implemented a novel dipeptide Andromeda score, which is the basis for a computationally efficient N-squared search engine. Additionally, partial scores summarize the evidence for the two constituents of the dipeptide individually. A posterior error probability (PEP) based on total and partial scores is used to control false discovery rates (FDRs). For MS-cleavable cross-linkers, a score of signature peaks is combined with the conventional Andromeda score on the cleavage products. The MaxQuant 3D peak detection was improved to ensure more accurate determination of the monoisotopic peak of isotope patterns for heavy molecules, which cross-linked peptides typically are. A wide selection of filtering parameters can replace the manual filtering of identifications, which is often necessary when using other pipelines. On benchmark data sets of synthetic peptides, MaxLynx outperforms all other tested software on data for both types of cross-linkers and on a proteome-wide data set of cross-linked Drosophila melanogaster cell lysate. The workflow also supports ion mobility-enhanced MS data. MaxLynx runs on Windows and Linux, contains an interactive viewer for displaying annotated cross-linked spectra, and is freely available at https://www.maxquant.org/.publishedVersio

Single Muscle Fiber Proteomics Reveals Fiber-Type-Specific Features of Human Muscle Aging

Skeletal muscle is a key tissue in human aging, which affects different muscle fiber types unequally. We developed a highly sensitive single muscle fiber proteomics workflow to study human aging and show that the senescence of slow and fast muscle fibers is characterized by diverging metabolic and protein quality control adaptations. Whereas mitochondrial content declines with aging in both fiber types, glycolysis and glycogen metabolism are upregulated in slow but downregulated in fast muscle fibers. Aging mitochondria decrease expression of the redox enzyme monoamine oxidase A. Slow fibers upregulate a subset of actin and myosin chaperones, whereas an opposite change happens in fast fibers. These changes in metabolism and sarcomere quality control may be related to the ability of slow, but not fast, muscle fibers to maintain their mass during aging. We conclude that single muscle fiber analysis by proteomics can elucidate pathophysiology in a sub-type-specific manner

Identification of signal peptide features for substrate specificity in human Sec62/Sec63-dependent ER protein import

In mammalian cells, one‐third of all polypeptides are integrated into the membrane or translocated into the lumen of the endoplasmic reticulum (ER) via the Sec61 channel. While the Sec61 complex facilitates ER import of most precursor polypeptides, the Sec61‐associated Sec62/Sec63 complex supports ER import in a substrate‐specific manner. So far, mainly posttranslationally imported precursors and the two cotranslationally imported precursors of ERj3 and prion protein were found to depend on the Sec62/Sec63 complex in vitro. Therefore, we determined the rules for engagement of Sec62/Sec63 in ER import in intact human cells using a recently established unbiased proteomics approach. In addition to confirming ERj3, we identified 22 novel Sec62/Sec63 substrates under these in vivo‐like conditions. As a common feature, those previously unknown substrates share signal peptides (SP) with comparatively longer but less hydrophobic hydrophobic region of SP and lower carboxy‐terminal region of SP (C‐region) polarity. Further analyses with four substrates, and ERj3 in particular, revealed the combination of a slowly gating SP and a downstream translocation‐disruptive positively charged cluster of amino acid residues as decisive for the Sec62/Sec63 requirement. In the case of ERj3, these features were found to be responsible for an additional immunoglobulin heavy‐chain binding protein (BiP) requirement and to correlate with sensitivity toward the Sec61‐channel inhibitor CAM741. Thus, the human Sec62/Sec63 complex may support Sec61‐channel opening for precursor polypeptides with slowly gating SPs by direct interaction with the cytosolic amino‐terminal peptide of Sec61α or via recruitment of BiP and its interaction with the ER‐lumenal loop 7 of Sec61α. These novel insights into the mechanism of human ER protein import contribute to our understanding of the etiology of SEC63‐linked polycystic liver disease

MaxDIA enables library-based and library-free data-independent acquisition proteomics

MaxDIA is a software platform for analyzing data-independent acquisition (DIA) proteomics data within the MaxQuant software environment. Using spectral libraries, MaxDIA achieves deep proteome coverage with substantially better coefficients of variation in protein quantification than other software. MaxDIA is equipped with accurate false discovery rate (FDR) estimates on both library-to-DIA match and protein levels, including when using whole-proteome predicted spectral libraries. This is the foundation of discovery DIA—hypothesis-free analysis of DIA samples without library and with reliable FDR control. MaxDIA performs three- or four-dimensional feature detection of fragment data, and scoring of matches is augmented by machine learning on the features of an identification. MaxDIA’s bootstrap DIA workflow performs multiple rounds of matching with increasing quality of recalibration and stringency of matching to the library. Combining MaxDIA with two new technologies—BoxCar acquisition and trapped ion mobility spectrometry—both lead to deep and accurate proteome quantification.publishedVersio

Phosphoproteomics analysis of a clinical mycobacterium tuberculosis Beijing isolate : expanding the mycobacterial phosphoproteome catalog

CITATION: Fortuin, S., et al. 2015. Phosphoproteomics analysis of a clinical mycobacterium tuberculosis beijing isolate : expanding the mycobacterial phosphoproteome catalog. Frontiers in Microbiology, 6:6, doi:10.3389/fmicb.2015.00006.The original publication is available at www.frontiersin.orgReversible protein phosphorylation, regulated by protein kinases and phosphatases, mediates a switch between protein activity and cellular pathways that contribute to a large number of cellular processes. The Mycobacterium tuberculosis genome encodes 11 Serine/Threonine kinases (STPKs) which show close homology to eukaryotic kinases. This study aimed to elucidate the phosphoproteomic landscape of a clinical isolate of M. tuberculosis. We performed a high throughput mass spectrometric analysis of proteins extracted from an early-logarithmic phase culture. Whole cell lysate proteins were processed using the filter-aided sample preparation method, followed by phosphopeptide enrichment of tryptic peptides by strong cation exchange (SCX) and Titanium dioxide (TiO2) chromatography. The MaxQuant quantitative proteomics software package was used for protein identification. Our analysis identified 414 serine/threonine/tyrosine phosphorylated sites, with a distribution of S/T/Y sites; 38% on serine, 59% on threonine and 3% on tyrosine; present on 303 unique peptides mapping to 214 M. tuberculosis proteins. Only 45 of the S/T/Y phosphorylated proteins identified in our study had been previously described in the laboratory strain H37Rv, confirming previous reports. The remaining 169 phosphorylated proteins were newly identified in this clinical M. tuberculosis Beijing strain. We identified 5 novel tyrosine phosphorylated proteins. These findings not only expand upon our current understanding of the protein phosphorylation network in clinical M. tuberculosis but the data set also further extends and complements previous knowledge regarding phosphorylated peptides and phosphorylation sites in M. tuberculosis.http://journal.frontiersin.org/article/10.3389/fmicb.2015.00006/fullPublisher's versio

First Community-Wide, Comparative Cross-Linking Mass Spectrometry Study

The number of publications in the field of chemical cross-linking combined with mass spectrometry (XL-MS) to derive constraints for protein three-dimensional structure modeling and to probe protein-protein interactions has increased during the last years. As the technique is now becoming routine for in vitro and in vivo applications in proteomics and structural biology there is a pressing need to define protocols as well as data analysis and reporting formats. Such consensus formats should become accepted in the field and be shown to lead to reproducible results. This first, community-based harmonization study on XL-MS is based on the results of 32 groups participating worldwide. The aim of this paper is to summarize the status quo of XL-MS and to compare and evaluate existing cross-linking strategies. Our study therefore builds the framework for establishing best practice guidelines to conduct cross-linking experiments, perform data analysis, and define reporting formats with the ultimate goal of assisting scientists to generate accurate and reproducible XL-MS results

Global Proteome Turnover Analyses of the Yeasts S. cerevisiae and S. pombe

Summary: How cells maintain specific levels of each protein and whether that control is evolutionarily conserved are key questions. Here, we report proteome-wide steady-state protein turnover rate measurements for the evolutionarily distant but ecologically similar yeasts, Saccharomyces cerevisiae and Schizosaccharomyces pombe. We find that the half-life of most proteins is much longer than currently thought and determined to a large degree by protein synthesis and dilution due to cell division. However, we detect a significant subset of proteins (∼15%) in both yeasts that are turned over rapidly. In addition, the relative abundances of orthologous proteins between the two yeasts are highly conserved across the 400 million years of evolution. In contrast, their respective turnover rates differ considerably. Our data provide a high-confidence resource for studying protein degradation in common yeast model systems. : Christiano et al. report protein turnover rates for S. cerevisiae and S. pombe. Overall, protein half-life is far less conserved than protein abundance. Protein turnover profiling of hrd1Δ, an endoplasmic-reticulum-associated degradation (ERAD)-defective mutant, identifies candidate substrates for this pathway in budding yeast