Tandem mass tags (TMT) are widely used in proteomics to simultaneously quantify multiple samples in a single experiment. The tags can be easily added to the primary amines of peptides/proteins through chemical reactions. In addition to amines, TMT reagents also partially react with the hydroxyl groups of serine, threonine, and tyrosine residues under alkaline conditions, which significantly compromises the analytical sensitivity and precision. Under alkaline conditions, reducing the TMT molar excess can partially mitigate overlabeling of histidine-free peptides, but has a limited effect on peptides containing histidine and hydroxyl groups. Here, we present a method under acidic conditions to suppress overlabeling while efficiently labeling amines, using only one-fifth of the TMT amount recommended by the manufacturer. In a deep-scale analysis of a yeast/human two-proteome sample, we systematically evaluated our method against the manufacturer’s method and a previously reported TMT-reduced method. Our method reduced overlabeled peptides by 9-fold and 6-fold, respectively, resulting in the substantial enhancement in peptide/protein identification rates. More importantly, the quantitative accuracy and precision were improved as overlabeling was reduced, endowing our method with greater statistical power to detect 42% and 12% more statistically significant yeast proteins compared to the standard and TMT-reduced methods, respectively. Mass spectrometric data have been deposited in the ProteomeXchange Consortium via the iProX partner repository with the data set identifier PXD047052.

The ProteomeXchange consortium has been set up to provide a single point of submission of MS proteomics data to the main existing proteomics repositories, and to encourage the data exchange between them for optimal data dissemination.

A data repository for proteomic data sets. The ProteomeExchange consortium, as a whole, aims to provide a coordinated submission of MS proteomics data to the main existing proteomics repositories, as well as to encourage optimal data dissemination. ProteomeXchange provides access to a number of public databases, and users can access and submit data sets to the consortium's PRIDE database and PASSEL/PeptideAtlas.

Mucin-domain glycoproteins expressed on cancer cell surfaces play central roles in cell adhesion, cancer progression, stem cell renewal, and immune evasion. Despite abundant evidence that mucin-domain glycoproteins are critical to the pathobiology of head and neck squamous cell carcinoma (HNSCC), our knowledge of the composition of that mucinome is grossly incomplete. Here, we utilized a catalytically inactive point mutant of the enzyme StcE (StcEE447D) to capture mucin-domain glycoproteins in head and neck cancer cell line lysates followed by their characterization using sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE), in-gel digestion, nano-liquid chromatography–tandem mass spectrometry (nLC-MS/MS), and enrichment analyses. We demonstrate the feasibility of this workflow for the study of mucin-domain glycoproteins in HNSCC, identify a set of mucin-domain glycoproteins common to multiple HNSCC cell lines, and report a subset of mucin-domain glycoproteins that are uniquely expressed in HSC-3 cells, a cell line derived from a highly aggressive metastatic tongue squamous cell carcinoma. This effort represents the first attempt to identify mucin-domain glycoproteins in HNSCC in an untargeted, unbiased analysis, paving the way for a more comprehensive characterization of the mucinome components that mediate aggressive tumor cell phenotypes. Data associated with this study have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD029420.

Credible detection and quantification of low abundance proteins from human blood plasma is a major challenge in precision medicine biomarker discovery when using mass spectrometry (MS). In this proof-of-concept study, we employed a mixture of selected recombinant proteins in DDA libraries to subsequently identify (not quantify) cancer-associated low abundance plasma proteins using SWATH/DIA. The exemplar DDA recombinant protein spectral library (rPSL) was derived from tryptic digestion of 36 recombinant human proteins that had been previously implicated as possible cancer biomarkers from both our own and other studies. The rPSL was then used to identify proteins from nondepleted colorectal cancer (CRC) EDTA plasmas by SWATH-MS. Most (32/36) of the proteins used in the rPSL were reliably identified from CRC plasma samples, including 8 proteins (i.e., BTC, CXCL10, IL1B, IL6, ITGB6, TGFα, TNF, TP53) not previously detected using high-stringency protein inference MS according to PeptideAtlas. The rPSL SWATH-MS protocol was compared to DDA-MS using MARS-depleted and postdigestion peptide fractionated plasmas (here referred to as a human plasma DDA library). Of the 32 proteins identified using rPSL SWATH, only 12 could be identified using DDA-MS. The 20 additional proteins exclusively identified using the rPSL SWATH approach were almost exclusively lower abundance (i.e.,

Integrated proteome resources center in China to accelerate data sharing in proteomics. Composed of data submission system and proteome database. Submission system is established under the guidance of data-sharing policy made by ProteomeXchange consortium. Registered users can submit their proteomic datasets to iProX in public or private modes. Once associated manuscript has been published, dataset becomes automatically public.

Dataset contains raw and preprocessed data for fluorescence and proteomic studies respectively. In each case, protein foldedness was probed using thiol reactivity. The raw mass spectrometry proteomics data have also been deposited to the ProteomeXchange Consortium via the PRIDE partner repository, with the dataset identifiers PXD022587 and PXD022640.

Dataset contains raw and preprocessed data for fluorescence and proteomic studies respectively. In each case, protein foldedness was probed using thiol reactivity. The raw mass spectrometry proteomics data have also been deposited to the ProteomeXchange Consortium via the PRIDE partner repository, with the dataset identifiers PXD033152.

Giardia duodenalis a species-complex of gastrointestinal protists, with assemblage A and B infective to humans. To date, post-genomic proteomics are largely derived from Assemblage A, biasing understanding of parasite biology. To address this gap, we quantitatively analysed the proteomes of trophozoites from the genome reference and two clinical Assemblage B isolates, revealing lower spectrum-to-peptide matches in non-reference isolates, resulting in significant losses in peptide and protein identifications, and indicating significant intra-assemblage variation. We also explored differential protein expression between in vitro cultured subpopulations putatively enriched for dividing and feeding cells, respectively. This data is an important proteomic baseline for Assemblage B, highlighting proteomic differences between physiological states, and unique differences relative to Assemblage A. The complete raw files and search results can be accessed via the ProteomeXchange Consortium (Vizcaino et al., 2013) via the PRIDE partner repository with the dataset identifier PXD007943.

Kinase enrichment utilizing broad-spectrum kinase inhibitors enables the identification of large proportions of the expressed kinome by mass spectrometry. However, the existing inhibitors are still inadequate in covering the entire kinome. Here, we identified a novel bisanilino pyrimidine, CTx-0294885, exhibiting inhibitory activity against a broad range of kinases in vitro, and further developed it into a Sepharose-supported kinase capture reagent. Use of a quantitative proteomics approach confirmed the selectivity of CTx-0294885-bound beads for kinase enrichment. Large-scale CTx-0294885-based affinity purification followed by LC–MS/MS led to the identification of 235 protein kinases from MDA-MB-231 cells, including all members of the AKT family that had not been previously detected by other broad-spectrum kinase inhibitors. Addition of CTx-0294885 to a mixture of three kinase inhibitors commonly used for kinase-enrichment increased the number of kinase identifications to 261, representing the largest kinome coverage from a single cell line reported to date. Coupling phosphopeptide enrichment with affinity purification using the four inhibitors enabled the identification of 799 high-confidence phosphosites on 183 kinases, ∼10% of which were localized to the activation loop, and included previously unreported phosphosites on BMP2K, MELK, HIPK2, and PRKDC. Therefore, CTx-0294885 represents a powerful new reagent for analysis of kinome signaling networks that may facilitate development of targeted therapeutic strategies. Proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the data set identifier PXD000239.

This is a public repository for proteomics data, containing identification data on mass spectrometric peptides and proteins, and post-translational modifications, in which data are supported by spectral evidence. PRIDE is a core member of the ProteomeXchange (PX) consortium.

Single colony metaproteomes of Trichodesmium from samples collected in North Atlantic surface waters during the R/V Atlantis cruise AT39-05 in March of 2018. These data were published in Held et al. (2020).

We performed proteomic analyses of human olfactory epithelial tissue to identify missing proteins using liquid chromatography–tandem mass spectrometry. Using a next-generation proteomic pipeline with a < 1.0% false discovery rate at the peptide and protein levels, we identified 3731 proteins, among which five were missing proteins (P0C7M7, P46721, P59826, Q658L1, and Q8N434). We validated the identified missing proteins using the corresponding synthetic peptides. No olfactory receptor (OR) proteins were detected in olfactory tissue, suggesting that detection of ORs would be very difficult. We also identified 49 and 50 alternative splicing variants mapped at the neXtProt and GENCODE databases, respectively, and 2000 additional single amino acid variants. This data set is available at the ProteomeXchange consortium via PRIDE repository (PXD010025).

A progressive loss of functional nephrons defines chronic kidney disease (CKD). Complications related to cardiovascular disease (CVD) are the principal causes of mortality in CKD; however, the acceleration of CVD in CKD remains unresolved. Our study used a complementary proteomic approach to assess mild and advanced CKD patients with different atherosclerosis stages and two groups of patients with different classical CVD progression but without renal dysfunction. We utilized a label-free approach based on LC-MS/MS and functional bioinformatic analyses to profile CKD and CVD leukocyte proteins. We revealed dysregulation of proteins involved in different phases of leukocytes’ diapedesis process that is very pronounced in CKD’s advanced stage. We also showed an upregulation of apoptosis-related proteins in CKD as compared to CVD. The differential abundance of selected proteins was validated by multiple reaction monitoring, ELISA, Western blotting, and at the mRNA level by ddPCR. An increased rate of apoptosis was then functionally confirmed on the cellular level. Hence, we suggest that the disturbances in leukocyte extravasation proteins may alter cell integrity and trigger cell death, as demonstrated by flow cytometry and microscopy analyses. Our proteomics data set has been deposited to the ProteomeXchange Consortium via the PRIDE repository with the data set identifier PXD018596.

The aim of this work was to test the effect of long-lasting heatwaves on the intertidal polychaete Hediste diversicolor (24 ºC vs 30 ºC for a month). We analysed the whole-body proteome and carried out fatty acid analysis after 28 days of exposure and then estimated cumulative survival and upper thermal tolerance limits (after 30 days of exposure). Worms' wet weight was also compared between temperatures (after 30 days) to understand if elevated temperature has effects on growth.

NOTE: The associated mass spectrometry proteomics dataset has been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD020635

Dataset associated to the paper DOI:10.1016/j.envres.2021.110885

Proteomic analysis of sensory organs such as the cochlea is challenging due to its small size and difficulties with membrane protein isolation. Mass spectrometry in conjunction with separation methods can provide a more comprehensive proteome, because of the ability to enrich protein samples, detect hydrophobic proteins, and identify low abundant proteins by reducing the proteome dynamic range. GELFrEE as well as different separation and digestion techniques were combined with FASP and nanoLC–MS/MS to obtain an in-depth proteome analysis of cochlear sensory epithelium from 30-day-old mice. Digestion with LysC/trypsin followed by SCX fractionation and multiple nanoLC–MS/MS analyses identified 3773 proteins with a 1% FDR. Of these, 694 protein IDs were in the plasmalemma. Protein IDs obtained by combining outcomes from GELFrEE/LysC/trypsin with GELFrEE/trypsin/trypsin generated 2779 proteins, of which 606 additional proteins were identified using the GELFrEE/LysC/trypsin approach. Combining results from the different techniques resulted in a total of 4620 IDs, including a number of previously unreported proteins. GO analyses showed high expression of binding and catalytic proteins as well as proteins associated with metabolism. The results show that the application of multiple techniques is needed to provide an exhaustive proteome of the cochlear sensory epithelium that includes many membrane proteins. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD000231.

This work was supported by project AquaMMIn [MAR-02.01.01-FEAMP-0038], co-funded by Portugal 2020 and the European Union through Mar2020, the Operational Programme (OP) for the European Maritime and Fisheries Fund (EMFF) in Portugal and by the Integrated Programme of SR&TD "SmartBioR - Smart Valorization of Endogenous Marine Biological Resources Under a Changing Climate" [Centro-01-0145-FEDER-000018], co-funded by Centro 2020 program, Portugal 2020, European Union, through the European Regional Development Fund. This work was also supported by L'Oreal/FCT/UNESCO through the prize L'Oreal Medals of Honour for Women in Science Portugal 2018 awarded to DM. Thanks are also due for the financial support to CESAM [UIDP/50017/2020+UIDB/50017/2020], QOPNA [FCT UID/QUI/00062/2019], LAQV/REQUIMTE [UIDB/50006/2020], and RNEM, Portuguese Mass Spectrometry Network [LISBOA-01-0145-FEDER-402-022125], to FCT/MEC through national funds [SFRH/BPD/117491/2016 and CEECIND/01250/2018 to DM], [PD/BD/127989/2016 to DJ] and to the co-funding by the FEDER, within the PT2020 Partnership Agreement and Compete 2020. The aim of this work was to test the effect of long-lasting heatwaves on the intertidal polychaete Hediste diversicolor (24 ºC vs 30 ºC for a month). We analysed the whole-body proteome and carried out fatty acid analysis after 28 days of exposure and then estimated cumulative survival and upper thermal tolerance limits (after 30 days of exposure). Worms' wet weight was also compared between temperatures (after 30 days) to understand if elevated temperature has effects on growth. NOTE: The associated mass spectrometry proteomics dataset has been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD020635 Dataset associated to the paper DOI:10.1016/j.envres.2021.110885

Description

Magnesium is an essential divalent metal that serves many cellular functions in growing cells. In Salmonella typhimurium, recent data from our collaborators of the PERIOMET ANR project showed the importance of magnesium in the ability of quiescent Salmonella cells to resume growth (Metaane et al. 2022 & 2023). During the study of magnesium and cobalt transport by CorA in Salmonella, three loci named corB, corC and corD, involved in resistance to cobalt and magnesium efflux have been discovered by Gibson et al. (1991). It was proposed that CorB, CorC and CorD contribute to the efflux of divalent cations by CorA, either by coupling the transport of cations by CorA to an energy source, or as a cation sensor for the CorA protein. The corB, corC and corD genes have not been further characterized in Salmonella and their function remains unclear to date. The corB, corC and corD genes correspond to the yfjD, ybeX and possibly apaG genes, respectively, in Salmonella serovar Typhimurium strain ATCC14028. Our collaborators have constructed deletions in these genes in order to study their physiological impact in Salmonella.

In the present study, we report the extrametabolome analysis by NMR of the WT and corC mutant in quiescent Salmonella cells after culture in presence of different magnesium concentrations.

In other related reports:

• Description of the construction of corC, corB and corD mutants and their resistance phenotype towards cobalt, nickel and magnesium toxicity. NOREL Françoise, MONTEIL Véronique, & METAANE Selma. (2023). Towards new elements involved in magnesium and cobalt trafficking in Salmonella serovar Typhimurium. Zenodo. https://doi.org/10.5281/zenodo.8086417
• The bacterial two hybrid system (BACTH) was used to find out potential physical interactions between CorA, CorB(YfjD), CorC(YbeX) and ApaG. NOREL F, METAANE S, & MONTEIL V. (2023). Detection of physical interactions between the magnesium transporter CorA and other Cor proteins using the bacterial two hybrid system (BACTH). Zenodo. https://doi.org/10.5281/zenodo.7994619
• Mass spectrometry proteomics was used to assess global effects of deletion of the corC gene on the Salmonella proteome. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD043903.

This work was supported by the French National Research Agency (ANR-19-CE44-0005-01, PERIOMET project).

References

• Gibson MM, Bagga DA, Miller CG, Maguire ME. Magnesium transport in Salmonella typhimurium: the influence of new mutations conferring Co2+ resistance on the CorA Mg2+ transport system. Molecular microbiology. 1991;5(11):2753-62.
• Metaane S, Monteil V, Ayrault S, Bordier L, Levi-Meyreuis C, Norel F. The stress sigma factor sigmaS/RpoS counteracts Fur repression of genes involved in iron and manganese metabolism and modulates the ionome of Salmonella enterica serovar Typhimurium. PloS one. 2022;17(3):e0265511.
• Metaane S, Monteil V, Douché T, Giai Gianetto Q, Matondo M, Maufrais C, Norel F. Loss of CorA, the primary magnesium transporter of Salmonella, is alleviated by MgtA and PhoP-dependent compensatory mechanisms. PloS one. 2023;18(9):e0291736.