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In the advanced stages, malignant melanoma (MM) has a very poor prognosis. Due to tremendous efforts in cancer research over the last 10 years, and the introduction of novel therapies such as targeted therapies and immunomodulators, the rather dark horizon of the median survival has dramatically changed from under 1 year to several years. With the advent of proteomics, deep-mining studies can reach low-abundant expression levels. The complexity of the proteome, however, still surpasses the dynamic range capabilities of current analytical techniques. Consequently, many predicted protein products with potential biological functions have not yet been verified in experimental proteomic data. This category of ‘missing proteins’ (MP) is comprised of all proteins that have been predicted but are currently unverified. As part of the initiative launched in 2016 in the USA, the European Cancer Moonshot Center has performed numerous deep proteomics analyses on samples from MM patients. In this study, nine MPs were clearly identified by mass spectrometry in MM metastases. Some MPs significantly correlated with proteins that possess identical PFAM structural domains; and other MPs were significantly associated with cancer-related proteins. This is the first study to our knowledge, where unknown and novel proteins have been annotated in metastatic melanoma tumour tissue.

The MM500 meta‐study aims to establish a knowledge basis of the tumor proteome to serve as a complement to genome and transcriptome studies. Somatic mutations and their effect on the transcriptome have been extensively characterized in melanoma. However, the effects of these genetic changes on the proteomic landscape and the impact on cellular processes in melanoma remain poorly understood. In this study, the quantitative mass‐spectrometry‐based proteomic analysis is interfaced with pathological tumor characterization, and associated with clinical data. The melanoma proteome landscape, obtained by the analysis of 505 well‐annotated melanoma tumor samples, is defined based on almost 16 000 proteins, including mutated proteoforms of driver genes. More than 50 million MS/MS spectra were analyzed, resulting in approximately 13,6 million peptide spectrum matches (PSMs). Altogether 13 176 protein‐coding genes, represented by 366 172 peptides, in addition to 52 000 phosphorylation sites, and 4 400 acetylation sites were successfully annotated. This data covers 65% and 74% of the predicted and identified human proteome, respectively. A high degree of correlation (Pearson, up to 0.54) with the melanoma transcriptome of the TCGA repository, with an overlap of 12 751 gene products, was found. Mapping of the expressed proteins with quantitation, spatiotemporal localization, mutations, splice isoforms, and PTM variants was proven not to be predicted by genome sequencing alone. The melanoma tumor molecular map was complemented by analysis of blood protein expression, including data on proteins regulated after immunotherapy. By adding these key proteomic pillars, the MM500 study expands the knowledge on melanoma disease., The MM500 meta‐study aims to establish a knowledge basis of the tumor proteome to serve as a complement to genome and transcriptome studies. The melanoma proteome landscape, obtained by the analysis of 505 well‐annotated melanoma tumor samples, is defined based on almost 16 000 proteins, including mutated proteoforms of driver genes. This data covers 65% and 74% of the predicted and identified human proteome, respectively.

Melanoma of the skin is the sixth most common type of cancer in Europe and accounts for 3.4% of all diagnosed cancers. More alarming is the degree of recurrence that occurs with approximately 20% of patients lethally relapsing following treatment. Malignant melanoma is a highly aggressive skin cancer and metastases rapidly extend to the regional lymph nodes (stage 3) and to distal organs (stage 4). Targeted oncotherapy is one of the standard treatment for progressive stage 4 melanoma, and BRAF inhibitors (e.g. vemurafenib, dabrafenib) combined with MEK inhibitor (e.g. trametinib) can effectively counter BRAFV600E-mutated melanomas. Compared to conventional chemotherapy, targeted BRAFV600E inhibition achieves a significantly higher response rate. After a period of cancer control, however, most responsive patients develop resistance to the therapy and lethal progression. The many underlying factors potentially causing resistance to BRAF inhibitors have been extensively studied. Nevertheless, the remaining unsolved clinical questions necessitate alternative research approaches to address the molecular mechanisms underlying metastatic and treatment-resistant melanoma. In broader terms, proteomics can address clinical questions far beyond the reach of genomics, by measuring, i.e. the relative abundance of protein products, post-translational modifications (PTMs), protein localisation, turnover, protein interactions and protein function. More specifically, proteomic analysis of body fluids and tissues in a given medical and clinical setting can aid in the identification of cancer biomarkers and novel therapeutic targets. Achieving this goal requires the development of a robust and reproducible clinical proteomic platform that encompasses automated biobanking of patient samples, tissue sectioning and histological examination, efficient protein extraction, enzymatic digestion, mass spectrometry-based quantitative protein analysis by label-free or labelling technologies and/or enrichment of peptides with specific PTMs. By combining data from, e.g. phosphoproteomics and acetylomics, the protein expression profiles of different melanoma stages can provide a solid framework for understanding the biology and progression of the disease. When complemented by proteogenomics, customised protein sequence databases generated from patient-specific genomic and transcriptomic data aid in interpreting clinical proteomic biomarker data to provide a deeper and more comprehensive molecular characterisation of cellular functions underlying disease progression. In parallel to a streamlined, patient-centric, clinical proteomic pipeline, mass spectrometry-based imaging can aid in interrogating the spatial distribution of drugs and drug metabolites within tissues at single-cell resolution. These developments are an important advancement in studying drug action and efficacy in vivo and will aid in the development of more effective and safer strategies for the treatment of melanoma. A collaborative effort of gargantuan proportions between academia and healthcare professionals has led to the initiation, establishment and development of a cutting-edge cancer research centre with a specialisation in melanoma and lung cancer. The primary research focus of the European Cancer Moonshot Lund Center is to understand the impact that drugs have on cancer at an individualised and personalised level. Simultaneously, the centre increases awareness of the relentless battle against cancer and attracts global interest in the exceptional research performed at the centre.

The MM500 study is an initiative to map the protein levels in malignant melanoma tumor samples, focused on in‐depth histopathology coupled to proteome characterization. The protein levels and localization were determined for a broad spectrum of diverse, surgically isolated melanoma tumors originating from multiple body locations. More than 15,500 proteoforms were identified by mass spectrometry, from which chromosomal and subcellular localization was annotated within both primary and metastatic melanoma. The data generated by global proteomic experiments covered 72% of the proteins identified in the recently reported high stringency blueprint of the human proteome. This study contributes to the NIH Cancer Moonshot initiative combining detailed histopathological presentation with the molecular characterization for 505 melanoma tumor samples, localized in 26 organs from 232 patients., The MM500 study is an initiative to map the protein levels in malignant melanoma tumor samples, focused on in‐depth histopathology coupled to proteome characterization. The protein levels and localization were determined for a broad spectrum of diverse melanoma tumors originating from multiple body locations. More than 15,500 proteoforms were identified by mass spectrometry, from which chromosomal and sub‐cellular localization was annotated within both primary and metastatic melanoma.

Urea-containing buffer solutions are generally used in proteomic studies to aid protein denaturation and solubilization during cell and tissue lysis. It is well-known, however, that urea can lead to carbamylation of peptides and proteins and, subsequently, incomplete digestion of proteins. By the use of cells and tissues that had been lysed with urea, different solution digestion strategies were quantitatively assessed. In comparison with traditional proteolysis at 37 °C, urea in-solution digestion performed at room temperature improved peptide and protein identification and quantitation and had a minimum impact on miscleavage rates. Furthermore, the signal intensities and the number of carbamylated and pyroglutamic acid-modified peptides decreased. Overall, this led to a reduction in the negative effects often observed for such modifications. Data are available via ProteomeXchange with identifier PXD009426.

Hypertrophic scars (HTS) and keloids are forms of aberrant cutaneous healing with excessive extracellular matrix (ECM) deposition. Current therapies still fall short and cause undesired effects. We aimed to thoroughly evaluate the ability of growth hormone releasing peptide 6 (GHRP6) to both prevent and reverse cutaneous fibrosis and to acquire the earliest proteome data supporting GHRP6's acute impact on aesthetic wound healing. Two independent sets of experiments addressing prevention and reversion effects were conducted on the classic HTS model in rabbits. In the prevention approach, the wounds were assigned to topically receive GHRP6, triamcinolone acetonide (TA), or vehicle (1% sodium carboxy methylcellulose [CMC]) from day 1 to day 30 post‐wounding. The reversion scheme was based on the infiltration of either GHRP6 or sterile saline in mature HTS for 4 consecutive weeks. The incidence and appearance of HTS were systematically monitored. The sub‐epidermal fibrotic core area of HTS was ultrasonographically determined, and the scar elevation index was calculated on haematoxylin/eosin‐stained, microscopic digitised images. Tissue samples were collected for proteomics after 1 hour of HTS induction and treatment with either GHRP6 or vehicle. GHRP6 prevented the onset of HTS without the untoward reactions induced by the first‐line treatment triamcinolone acetonide (TA); however, it failed to significantly reverse mature HTS. The preliminary proteomic study suggests that the anti‐fibrotic preventing effect exerted by GHRP6 depends on different pathways involved in lipid metabolism, cytoskeleton arrangements, epidermal cells’ differentiation, and ECM dynamics. These results enlighten the potential success of GHRP6 as one of the incoming alternatives for HTS prevention.

The protein composition of an Outer Membrane Vesicle (OMV) preparation that constitutes the active pharmaceutical ingredient of VA-MENGOC-BC®, an effective vaccine against Neisseria meningitidis serogroups B, and C is presented. This preparation has a high lipid content and five abundant membrane proteins (FetA, PorA, PorB, RmpM, and Opc), constituting approximately 70% of the total protein mass. The protein composition was determined by combining the use of the Hexapeptide Ligand Library and an orthogonal tandem fractionation of tryptic peptides by reverse-phase chromatography at alkaline and acid pH. This approach equalizes the concentration of tryptic peptides derived from low- and high-abundance proteins as well as considerably simplifying the number of peptides analyzed by LC-MS/MS, enhancing the possibility of identifying low-abundance species. Fifty-one percent of the proteins originally annotated as membrane proteins in the genome of the MC58 strain were identified. One hundred and sixty-eight low-abundance cytosolic proteins presumably occluded within OMV were also identified. Four (NadA, NUbp, GNA2091, and fHbp), out of the five antigens constituting the Bexsero® vaccine, were detected in this OMV preparation. In particular, fHbp is also the active principle of the Trumenba® vaccine developed by Pfizer. The HpuA and HpuB gene products (not annotated in the MC58 genome) were identified in the CU385 strain, a clinical isolate that is used to produce this OMV. Considering the proteins identified here and previous work done by our group, the protein catalogue of this OMV preparation was extended to 266 different protein species.

In comparison to other human cancer types, malignant melanoma exhibits the greatest amount of heterogeneity. After DNA-based detection of the BRAF V600E mutation in melanoma patients, targeted inhibitor treatment is the current recommendation. This approach, however, does not take the abundance of the therapeutic target, i.e., the B-raf V600E protein, into consideration. As shown by immunohistochemistry, the protein expression profiles of metastatic melanomas clearly reveal the existence of inter- and intra-tumor variability. Nevertheless, the technique is only semi-quantitative. To quantitate the mutant protein there is a fundamental need for more precise techniques that are aimed at defining the currently non-existent link between the levels of the target protein and subsequent drug efficacy. Using cutting-edge mass spectrometry combined with DNA and mRNA sequencing, the mutated B-raf protein within metastatic tumors was quantitated for the first time. B-raf V600E protein analysis revealed a subjacent layer of heterogeneity for mutation-positive metastatic melanomas. These were characterized into two distinct groups with different tumor morphologies, protein profiles and patient clinical outcomes. This study provides evidence that a higher level of expression in the mutated protein is associated with a more aggressive tumor progression. Our study design, comprised of surgical isolation of tumors, histopathological characterization, tissue biobanking, and protein analysis, may enable the eventual delineation of patient responders/non-responders and subsequent therapy for malignant melanoma.

In comparison to other human cancer types, malignant melanoma exhibits the greatest amount of heterogeneity. After DNA-based detection of the BRAF V600E mutation in melanoma patients, targeted inhibitor treatment is the current recommendation. This approach, however, does not take the abundance of the therapeutic target, i.e., the B-raf V600E protein, into consideration. As shown by immunohistochemistry, the protein expression profiles of metastatic melanomas do clearly reveal the existence of inter- and intra-tumor variability. Nevertheless, the technique is only semi-quantitative. To quantitate the mutant protein there is a fundamental need for more precise techniques that are aimed at defining the currently non-existent link between the levels of the target protein and subsequent drug efficacy. Using cutting-edge mass spectrometry combined with DNA and mRNA sequencing, the mutated B-raf protein within metastatic tumors was quantitated for the first time. B-raf V600E protein analysis revealed a subjacent layer of heterogeneity for mutation-positive metastatic melanomas. These were characterized into two distinct groups with different tumor morphologies, protein profiles and patient clinical outcomes. This study provides evidence that a higher level of expression for the mutated protein is associated with a more aggressive tumor progression. Our study design that is comprised of surgical isolation of tumors, histopathological characterization, tissue biobanking, and protein analysis may enable the eventual delineation of patient responders/non-responders and the subsequent therapy of malignant melanoma.

Metastatic melanoma is one of the most common deadly cancers, and robust biomarkers are still needed, e.g. to predict survival and treatment efficiency. Here, protein expression analysis of one hundred eleven melanoma lymph node metastases using high resolution mass spectrometry is coupled with in-depth histopathology analysis, clinical data and genomics profiles. This broad view of protein expression allowed to identify novel candidate protein markers that improved prediction of survival in melanoma patients. Some of the prognostic proteins have not been reported in the context of melanoma before, and few of them exhibit unexpected relationship to survival, which likely reflects the limitations of current knowledge on melanoma and shows the potential of proteomics in clinical cancer research.

Large cohorts of carefully collected clinical tissue materials play a central role in acquiring sufficient depth and statistical power to discover disease-related mechanisms and biomarkers of clinical significance. Manual preparation of such large sample cohorts requires experienced laboratory personnel. This carries other possible downsides such as low throughput, high risk of errors, and low reproducibility. In this work, three automated technologies for high-throughput proteomics of frozen sectioned tissues were compared. The instruments evaluated included the Bioruptor for tissue disruption and protein extraction; the Barocycler, which is able to disrupt tissues and digest the proteins; and the AssayMAP Bravo, a microchromatography platform for protein digestion, peptide desalting, and fractionation. Wide varieties of tissue samples from rat spleen, malignant melanoma, and pancreatic tumors were used for the assessment. The three instruments displayed reproducible and consistent results, as was proven by high correlations and low coefficients of variation between technical replicates and even more importantly, between replicates that were processed in different batches or at different time points. The results from this study allowed us to integrate these technologies into an automated sample preparation workflow for large-scale proteomic studies that are currently ongoing. Data are available via ProteomeXchange with identifiers PXD010296 and PXD011295.

There is an increasing global interest to support research areas that can assist in understanding disease and improving patient care. The National Cancer Institute (NIH) has identified precision medicine-based approaches as key research strategies to expedite advances in cancer research. The Cancer Moonshot program (https://www.cancer.gov/research/key-initiatives/moonshot-cancer-initiative) is the largest cancer program of all time, and has been launched to accelerate cancer research that aims to increase the availability of therapies to more patients and, ultimately, to eradicate cancer. Mass spectrometry-based proteomics has been extensively used to study the molecular mechanisms of cancer, to define molecular subtypes of tumors, to map cancer-associated protein interaction networks and post-translational modifications, and to aid in the development of new therapeutics and new diagnostic and prognostic tests. To establish the basis for our melanoma studies, we have established the Southern Sweden Malignant Melanoma Biobank. Tissues collected over many years have been accurately characterized with respect to the tumor and patient information. The extreme variability displayed in the protein profiles and the detection of missense mutations has confirmed the complexity and heterogeneity of the disease. It is envisaged that the combined analysis of clinical, histological, and proteomic data will provide patients with a more personalized medical treatment. With respect to disease presentation, targeted treatment and medical mass spectrometry analysis and imaging, this overview report will outline and summarize the current achievements and status within malignant melanoma. We present data generated by our cancer research center in Lund, Sweden, where we have built extensive capabilities in biobanking, proteogenomics, and patient treatments over an extensive time period. © 2018, The Author(s)., Funding information This study was supported by a joint grant from Berta Kamprad Foundation, and grants from the National Research Foundation of Korea, funded by the government of Republic of Korea (MSIP; 2012M3A9D1054520, 2015K1A1A2028365, 2015M3A9C4076 321), European Union’s Horizon 2020, Interreg. Program: NYPS 20200407, BReproUnion^, and European Union Regional Development Fund, Interreg. Oresund, BMAX4ESSFUN, LU-062^.

Here we describe an integrated approach for the selective separation of peptides from complex mixtures using strong cation-exchange chromatography. The procedure exploits the charge differences produced by reversible modification of primary amino groups in peptides, enabling their separation into three major fractions: 1) neutral peptides 2) peptides with one positive charge and 3) peptides with 2 or more positive charges. The procedure demonstrated an excellent selectivity which allowed restricted MS/MS ion searches with peptide-centric databases.

Neisseria meningitidis is a Gram-negative bacterium responsible for significant mortality worldwide. While effective polysaccharides-based vaccines exist against serogroups A, C, W135, and Y, no similar vaccine is suitable for children under 4 years against disease caused by serogroup B strains. Therefore, major vaccine efforts against this serogroup are based on outer membrane vesicles (OMVs), containing major outer membrane proteins. The OMV-based vaccine produced by the Finlay Institute in Cuba (VA-MENGOC-BC®) contributed to the rapid decline of the epidemic in this Caribbean island. While the content of major proteins in this vaccine has been discussed, no detailed work of an outer membrane proteomic map of this, or any other, commercially available OMV-derived product has been published so far. Since OMVs exhibit a large bias toward a few major proteins and usually contain a high content of lipids, establishing the adequate conditions for high resolution, 2-DE of this kind of preparation was definitely a technical challenge. In this work, 2-DE and MS have been used to generate a proteomic map of this product, detailing the presence of 31 different proteins, and it allows the identification of new putative protective protein components it contains.

In this work, a method devised for the selective isolation of multiply-charged peptide applied to a complex protein mixture was evaluated for the first time using a mass spectrometer with low resolution (LTQ). In this procedure, all primary amino groups of tryptic peptides derived from human liver tissue interstitial fluid (TIF) are blocked, restricting their positive charge, at acidic pH, to the presence of histidine and arginine residues. After strong cation exchange chromatography, multiply-charged peptides (#R + #H > 1) are retained in the column and separated with high selectivity from singly (#R + #H = 1) and neutral peptides (#R + #H = 0) which are collected together in the flow-through. Using liquid chromatography electrospray ionization tandem mass spectrometry analysis the retained fraction displayed a 95% enrichment of multiply charged peptides while in the flow-through; only 4% of multiply-charged peptides were identified.

Peptide sequence matching algorithms used for peptide identification by tandem mass spectrometry (MS/MS) enumerate theoretical peptides from the database, predict their fragment ions, and match them to the experimental MS/MS spectra. Here, we present an approach for scoring MS/MS identifications based on the high mass accuracy matching of precursor ions, the identification of a high intensity b1 fragment ion, and partial sequence tags from phenylthiocarbamoyl-derivatized peptides. This derivatization process boosts the b1 fragment ion signal, which turns it into a powerful feature for peptide identification. We demonstrate the effectiveness of our scoring system by implementing it on a computational tool called "HI-bone" and by identifying mass spectra of an Escherichia coli sample acquired on an Orbitrap Velos instrument using Higher-energy C-trap dissociation. Following this strategy, we identified 1614 peptide spectrum matches with a peptide false discovery rate (FDR) below 1%. These results were significantly higher than those from Mascot and SEQUEST using a similar FDR.

A combination of antiviral drugs known as antiretroviral therapy (ART) has shown effectiveness against the human immunodeficiency virus (HIV). ART has markedly decreased mortality and morbidity among HIV-infected patients, having even reduced HIV transmission. However, an important current disadvantage, resistance development, remains to be solved. Hope is focused on developing drugs against cellular targets. This strategy is expected to prevent the emergence of viral resistance. In this study, using a comparative proteomic approach in MT4 cells treated with an anti-HIV leukocyte extract, we identified vimentin, a molecule forming intermediate filaments in the cell, as a possible target against HIV infection. We demonstrated a strong reduction of an HIV-1 based lentivirus expressing the enhanced green fluorescent protein (eGFP) in vimentin knockdown cells, and a noteworthy decrease of HIV-1 capsid protein antigen (CAp24) in those cells using a multiround infectivity assay. Electron micrographs showed changes in the structure of intermediate filaments when MT4 cells were treated with an anti-HIV leukocyte extract. Changes in the structure of intermediate filaments were also observed in vimentin knockdown MT4 cells. A synthetic peptide derived from a cytoskeleton protein showed potent inhibitory activity on HIV-1 infection, and low cytotoxicity. Our data suggest that vimentin can be a suitable target to inhibit HIV-1.

Here we demonstrate the usefulness of peptide fractionation by SDS-free polyacrylamide gel electrophoresis and its applicability to proteomics studies. In the absence of SDS, the driving force for the electrophoretic migration toward the anode is supplied by negatively charged acidic amino acid residues and other residues as phosphate, sulfate and sialic acid, while the resulting mobility depends on both the charge and the molecular mass of the peptides. A straightforward method was achieved for SDS-PAGE of proteins, enzyme digestion, peptide transfer and fractionation by SDS-free PAGE, which was named dual-fractionation polyacrylamide gel electrophoresis (DF-PAGE). This method increases the number of identified proteins 2.5-fold with respect to the proteins identified after direct analysis, and more than 80% of assigned peptides were found in unique SDS-free gel slices. A vast majority of identified peptides (93%) have p I values below 7.0, and 7% have p I values between 7.0 and 7.35. Peptide digests that were derived from complex protein mixtures were in consequence simplified as peptides that are positively charged are not recovered in the present conditions. The analysis of a membrane protein extract from Neisseria meningitidis by this approach allowed the identification of 97 proteins, including low-abundance components.

Tryptic digestion of biotinylated Lys-C peptides followed by affinity chromatography allows the selective isolation of lysine-free tryptic peptides delimited by arginine residues (RRnK peptides). In silico analysis revealed that RRnK peptides represent 87% of the whole proteomes and their specific isolation simplifies the complex peptide mixture (5 peptides per protein). The good recoveries and high selectivity obtained in the isolation of RRnK peptides anticipate the applicability of this method in 2DE-free quantitative proteome analyses. Keywords: proteomics • selective-peptide-isolation • protein-identification • affinity chromatography • mass spectrometry