A region-resolved proteomic map of the human brain enabled by high-throughput proteomics.

Substantial efforts are underway to deepen our understanding of human brain morphology, structure, and function using high-resolution imaging as well as high-content molecular profiling technologies. The current work adds to these approaches by providing a comprehensive and quantitative protein expression map of 13 anatomically distinct brain regions covering more than 11,000 proteins. This was enabled by the optimization, characterization, and implementation of a high-sensitivity and high-throughput microflow liquid chromatography timsTOF tandem mass spectrometry system (LC-MS/MS) capable of analyzing more than 2,000 consecutive samples prepared from formalin-fixed paraffin embedded (FFPE) material. Analysis of this proteomic resource highlighted brain region-enriched protein expression patterns and functional protein classes, protein localization differences between brain regions and individual markers for specific areas. To facilitate access to and ease further mining of the data by the scientific community, all data can be explored online in a purpose-built R Shiny app (https://brain-region-atlas.proteomics.ls.tum.de).

Proximity labeling of host factor ANXA3 in HCV infection reveals a novel LARP1 function in viral entry.

Hepatitis C virus (HCV) infection is tightly connected to the lipid metabolism with lipid droplets (LDs) serving as assembly sites for progeny virions. A previous LD proteome analysis identified annexin A3 (ANXA3) as an important HCV host factor that is enriched at LDs in infected cells and required for HCV morphogenesis. To further characterize ANXA3 function in HCV, we performed proximity labeling using ANXA3-BioID2 as bait in HCV-infected cells. Two of the top proteins identified proximal to ANXA3 during HCV infection were the La-related protein 1 (LARP1) and the ADP ribosylation factor-like protein 8B (ARL8B), both of which have been previously described to act in HCV particle production. In follow-up experiments, ARL8B functioned as a pro-viral HCV host factor without localizing to LDs and thus likely independent of ANXA3. In contrast, LARP1 interacts with HCV core protein in an RNA-dependent manner and is translocated to LDs by core protein. Knockdown of LARP1 decreased HCV spreading without altering HCV RNA replication or viral titers. Unexpectedly, entry of HCV particles and E1/E2-pseudotyped lentiviral particles was reduced by LARP1 depletion, whereas particle production was not altered. Using a recombinant vesicular stomatitis virus (VSV)ΔG entry assay, we showed that LARP1 depletion also decreased entry of VSV with VSV, MERS, and CHIKV glycoproteins. Therefore, our data expand the role of LARP1 as an HCV host factor that is most prominently involved in the early steps of infection, likely contributing to endocytosis of viral particles through the pleiotropic effect LARP1 has on the cellular translatome.

Rhizochalinin Exhibits Anticancer Activity and Synergizes with EGFR Inhibitors in Glioblastoma In Vitro Models.

Rhizochalinin (Rhiz) is a recently discovered cytotoxic sphingolipid synthesized from the marine natural compound rhizochalin. Previously, Rhiz demonstrated high in vitro and in vivo efficacy in various cancer models. Here, we report Rhiz to be highly active in human glioblastoma cell lines as well as in patient-derived glioma-stem like neurosphere models. Rhiz counteracted glioblastoma cell proliferation by inducing apoptosis, G2/M-phase cell cycle arrest, and inhibition of autophagy. Proteomic profiling followed by bioinformatic analysis suggested suppression of the Akt pathway as one of the major biological effects of Rhiz. Suppression of Akt as well as IGF-1R and MEK1/2 kinase was confirmed in Rhiz-treated GBM cells. In addition, Rhiz pretreatment resulted in a more pronounced inhibitory effect of γ-irradiation on the growth of patient-derived glioma-spheres, an effect to which the Akt inhibition may also contribute decisively. In contrast, EGFR upregulation, observed in all GBM neurospheres under Rhiz treatment, was postulated to be a possible sign of incipient resistance. In line with this, combinational therapy with EGFR-targeted tyrosine kinase inhibitors synergistically increased the efficacy of Rhiz resulting in dramatic inhibition of GBM cell viability as well as a significant reduction of neurosphere size in the case of combination with lapatinib. Preliminary in vitro data generated using a parallel artificial membrane permeability (PAMPA) assay suggested that Rhiz cannot cross the blood brain barrier and therefore alternative drug delivery methods should be used in the further in vivo studies. In conclusion, Rhiz is a promising new candidate for the treatment of human glioblastoma, which should be further developed in combination with EGFR inhibitors.

ELF5 modulates the estrogen receptor cistrome in breast cancer.

Acquired resistance to endocrine therapy is responsible for half of the therapeutic failures in the treatment of breast cancer. Recent findings have implicated increased expression of the ETS transcription factor ELF5 as a potential modulator of estrogen action and driver of endocrine resistance, and here we provide the first insight into the mechanisms by which ELF5 modulates estrogen sensitivity. Using chromatin immunoprecipitation sequencing we found that ELF5 binding overlapped with FOXA1 and ER at super enhancers, enhancers and promoters, and when elevated, caused FOXA1 and ER to bind to new regions of the genome, in a pattern that replicated the alterations to the ER/FOXA1 cistrome caused by the acquisition of resistance to endocrine therapy. RNA sequencing demonstrated that these changes altered estrogen-driven patterns of gene expression, the expression of ER transcription-complex members, and 6 genes known to be involved in driving the acquisition of endocrine resistance. Using rapid immunoprecipitation mass spectrometry of endogenous proteins, and proximity ligation assays, we found that ELF5 interacted physically with members of the ER transcription complex, such as DNA-PKcs. We found 2 cases of endocrine-resistant brain metastases where ELF5 levels were greatly increased and ELF5 patterns of gene expression were enriched, compared to the matched primary tumour. Thus ELF5 alters ER-driven gene expression by modulating the ER/FOXA1 cistrome, by interacting with it, and by modulating the expression of members of the ER transcriptional complex, providing multiple mechanisms by which ELF5 can drive endocrine resistance.

Comparative Assessment of Quantification Methods for Tumor Tissue Phosphoproteomics.

With increasing sensitivity and accuracy in mass spectrometry, the tumor phosphoproteome is getting into reach. However, the selection of quantitation techniques best-suited to the biomedical question and diagnostic requirements remains a trial and error decision as no study has directly compared their performance for tumor tissue phosphoproteomics. We compared label-free quantification (LFQ), spike-in-SILAC (stable isotope labeling by amino acids in cell culture), and tandem mass tag (TMT) isobaric tandem mass tags technology for quantitative phosphosite profiling in tumor tissue. Compared to the classic SILAC method, spike-in-SILAC is not limited to cell culture analysis, making it suitable for quantitative analysis of tumor tissue samples. TMT offered the lowest accuracy and the highest precision and robustness toward different phosphosite abundances and matrices. Spike-in-SILAC offered the best compromise between these features but suffered from a low phosphosite coverage. LFQ offered the lowest precision but the highest number of identifications. Both spike-in-SILAC and LFQ presented susceptibility to matrix effects. Match between run (MBR)-based analysis enhanced the phosphosite coverage across technical replicates in LFQ and spike-in-SILAC but further reduced the precision and robustness of quantification. The choice of quantitative methodology is critical for both study design such as sample size in sample groups and quantified phosphosites and comparison of published cancer phosphoproteomes. Using ovarian cancer tissue as an example, our study builds a resource for the design and analysis of quantitative phosphoproteomic studies in cancer research and diagnostics.

Murine cytomegaloviruses m139 targets DDX3 to curtail interferon production and promote viral replication.

Cytomegaloviruses (CMV) infect many different cell types and tissues in their respective hosts. Monocytes and macrophages play an important role in CMV dissemination from the site of infection to target organs. Moreover, macrophages are specialized in pathogen sensing and respond to infection by secreting cytokines and interferons. In murine cytomegalovirus (MCMV), a model for human cytomegalovirus, several genes required for efficient replication in macrophages have been identified, but their specific functions remain poorly understood. Here we show that MCMV m139, a gene of the conserved US22 gene family, encodes a protein that interacts with the DEAD box helicase DDX3, a protein involved in pathogen sensing and interferon (IFN) induction, and the E3 ubiquitin ligase UBR5. DDX3 and UBR5 also participate in the transcription, processing, and translation of a subset of cellular mRNAs. We show that m139 inhibits DDX3-mediated IFN-α and IFN-ß induction and is necessary for efficient viral replication in bone-marrow derived macrophages. In vivo, m139 is crucial for viral dissemination to local lymph nodes and to the salivary glands. An m139-deficient MCMV also replicated to lower titers in SVEC4-10 endothelial cells. This replication defect was not accompanied by increased IFN-ß transcription, but was rescued by knockout of either DDX3 or UBR5. Moreover, m139 co-localized with DDX3 and UBR5 in viral replication compartments in the cell nucleus. These results suggest that m139 inhibits DDX3-mediated IFN production in macrophages and antagonizes DDX3 and UBR5-dependent functions related to RNA metabolism in endothelial cells.

Invariable stoichiometry of ribosomal proteins in mouse brain tissues with aging.

Across phyla, the ribosomes-the central molecular machines for translation of genetic information-exhibit an overall preserved architecture and a conserved functional core. The natural heterogeneity of the ribosome periodically phases a debate on their functional specialization and the tissue-specific variations of the ribosomal protein (RP) pool. Using sensitive differential proteomics, we performed a thorough quantitative inventory of the protein composition of ribosomes from 3 different mouse brain tissues, i.e., hippocampus, cortex, and cerebellum, across various ages, i.e., juvenile, adult, and middle-aged mouse groups. In all 3 brain tissues, in both monosomal and polysomal ribosome fractions, we detected an invariant set of 72 of 79 core RPs, RACK1 and 2 of the 8 RP paralogs, the stoichiometry of which remained constant across different ages. The amount of a few RPs punctually varied in either one tissue or one age group, but these fluctuations were within the tight bounds of the measurement noise. Further comparison with the ribosomes from a high-metabolic-rate organ, e.g., the liver, revealed protein composition identical to that of the ribosomes from the 3 brain tissues. Together, our data show an invariant protein composition of ribosomes from 4 tissues across different ages of mice and support the idea that functional heterogeneity may arise from factors other than simply ribosomal protein stoichiometry.

Salvage Chemotherapy with Cisplatin, Ifosfamide, and Paclitaxel in Aggressive Variant of Metastatic Castration-Resistant Prostate Cancer.

Significant progress has been achieved in the treatment of metastatic castration-resistant prostate cancer (mCRPC). However, results in patients with aggressive variant prostate cancer (AVPC) have been disappointing. Here, we report retrospectively collected data from intensively pretreated AVPC patients (n = 17; 88.2% visceral metastases; 82% elevation of neuroendocrine markers) treated with salvage chemotherapy consisting of cisplatin, ifosfamide, and paclitaxel (TIP). At the interim analysis, 60% of patients showed radiographic response or stable disease (PFS = 2.5 months; OS = 6 months). In men who responded to chemotherapy, an OS > 15 months was observed. Preclinical analyses confirmed the high activity of the TIP regimen, especially in docetaxel-resistant prostate cancer cells. This effect was primarily mediated by increased cisplatin sensitivity in the emergence of taxane resistance. Proteomic and functional analyses identified a lower DNA repair capacity and cell cycle machinery deficiency to be causative. In contrast, paclitaxel showed inconsistent effects, partially antagonizing cisplatin and ifosfamide in some AVPC models. Consequently, paclitaxel has been excluded from the TIP combination for future patients. In summary, we report for the first time the promising efficacy of TIP as salvage therapy in AVPC. Our preclinical data indicate a pivotal role for cisplatin in overcoming docetaxel resistance.

Proteome analysis of human adipocytes identifies depot-specific heterogeneity at metabolic control points.

Adipose tissue is a primary regulator of energy balance and metabolism. The distribution of adipose tissue depots is of clinical interest because the accumulation of upper-body subcutaneous (ASAT) and visceral adipose tissue (VAT) is associated with cardiometabolic diseases, whereas lower-body glutealfemoral adipose tissue (GFAT) appears to be protective. There is heterogeneity in morphology and metabolism of adipocytes obtained from different regions of the body, but detailed knowledge of the constituent proteins in each depot is lacking. Here, we determined the human adipocyte proteome from ASAT, VAT, and GFAT using high-resolution Sequential Window Acquisition of all Theoretical (SWATH) mass spectrometry proteomics. We quantified 4,220 proteins in adipocytes, and 2,329 proteins were expressed in all three adipose depots. Comparative analysis revealed significant differences between adipocytes from different regions (6% and 8% when comparing VAT vs. ASAT and GFAT, 3% when comparing the subcutaneous adipose tissue depots, ASAT and GFAT), with marked differences in proteins that regulate metabolic functions. The VAT adipocyte proteome was overrepresented with proteins of glycolysis, lipogenesis, oxidative stress, and mitochondrial dysfunction. The GFAT adipocyte proteome predicted the activation of peroxisome proliferator-activated receptor α (PPARα), fatty acid, and branched-chain amino acid (BCAA) oxidation, enhanced tricarboxylic acid (TCA) cycle flux, and oxidative phosphorylation, which was supported by metabolomic data obtained from adipocytes. Together, this proteomic analysis provides an important resource and novel insights that enhance the understanding of metabolic heterogeneity in the regional adipocytes of humans.NEW & NOTEWORTHY Adipocyte metabolism varies depending on anatomical location and the adipocyte protein composition may orchestrate this heterogeneity. We used SWATH proteomics in patient-matched human upper- (visceral and subcutaneous) and lower-body (glutealfemoral) adipocytes and detected 4,220 proteins and distinguishable regional proteomes. Upper-body adipocyte proteins were associated with glycolysis, de novo lipogenesis, mitochondrial dysfunction, and oxidative stress, whereas lower-body adipocyte proteins were associated with enhanced PPARα activation, fatty acid, and BCAA oxidation, TCA cycle flux, and oxidative phosphorylation.

Cell-autonomous hepatocyte-specific GP130 signaling is sufficient to trigger a robust innate immune response in mice.

BACKGROUND & AIMS: Interleukin (IL)-6 cytokine family members contribute to inflammatory and regenerative processes. Engagement of the signaling receptor subunit gp130 is common to almost all members of the family. In the liver, all major cell types respond to IL-6-type cytokines, making it difficult to delineate cell type-specific effects. We therefore generated mouse models for liver cell type-specific analysis of IL-6 signaling. METHODS: We produced mice with a Cre-inducible expression cassette encoding a designed pre-dimerized constitutive active gp130 variant. We bred these mice to different Cre-drivers to induce transgenic gp130 signaling in distinct liver cell types: hepatic stellate cells, cholangiocytes/liver progenitor cells or hepatocytes. We phenotyped these mice using multi-omics approaches, immunophenotyping and a bacterial infection model. RESULTS: Hepatocyte-specific gp130 activation led to the upregulation of innate immune system components, including acute-phase proteins. Consequently, we observed peripheral mobilization and recruitment of myeloid cells to the liver. Hepatic myeloid cells, including liver-resident Kupffer cells were instructed to adopt a bactericidal phenotype which ultimately conferred enhanced resistance to bacterial infection in these mice. We demonstrate that persistent hepatocyte-specific gp130 activation resulted in amyloid A amyloidosis in aged mice. In contrast, we did not observe overt effects of hepatic stellate cell- or cholangiocyte/liver progenitor cell-specific transgenic gp130 signaling. CONCLUSIONS: Hepatocyte-specific gp130 activation alone is sufficient to trigger a robust innate immune response in the absence of NF-κB activation. We therefore conclude that gp130 engagement, e.g. by IL-6 trans-signaling, represents a safe-guard mechanism in innate immunity. LAY SUMMARY: Members of the interleukin-6 cytokine family signal via the receptor subunit gp130 and are involved in multiple processes in the liver. However, as several liver cell types respond to interleukin-6 family cytokines, it is difficult to delineate cell type-specific effects. Using a novel mouse model, we provide evidence that hepatocyte-specific gp130 activation is sufficient to trigger a robust systemic innate immune response.

Investigation of the Proteomes of the Truffles Tuber albidum pico, T. aestivum, T. indicum, T. magnatum, and T. melanosporum.

Truffles of the Tuber species are known as expensive foods, mainly for their distinct aroma and taste. This high price makes them a profitable target of food fraud, e.g., the misdeclaration of cheaper truffle species as expensive ones. While many studies investigated truffles on the metabolomic level or the volatile organic compounds extruded by them, research at the proteome level as a phenotype determining basis is limited. In this study, a bottom-up proteomic approach based on LC-MS/MS measurements in data-independent acquisition mode was performed to analyze the truffle species Tuber aestivum, Tuber albidum pico, Tuber indicum, Tuber magnatum, and Tuber melanosporum, and a protein atlas of the investigated species was obtained. The yielded proteomic fingerprints are unique for each of the of the five truffle species and can now be used in case of suspected food fraud. First, a comprehensive spectral library containing 9000 proteins and 50,000 peptides was generated by two-dimensional liquid chromatography coupled to tandem mass spectrometry (2D-LC-MS/MS). Then, samples of the truffle species were analyzed in data-independent acquisition (DIA) proteomics mode yielding 2715 quantified proteins present in all truffle samples. Individual species were clearly distinguishable by principal component analysis (PCA). Quantitative proteome fingerprints were generated from 2066 ANOVA significant proteins, and side-by-side comparisons of truffles were done by T-tests. A further aim of this study was the annotation of functions for the identified proteins. For Tuber magnatum and Tuber melanosporum conclusive links to their superior aroma were found by enrichment of proteins responsible for sulfur-metabolic processes in comparison with other truffles. The obtained data in this study may serve as a reference library for food analysis laboratories in the future to tackle food fraud by misdeclaration of truffles. Further identified proteins with their corresponding abundance values in the different truffle species may serve as potential protein markers in the establishment of targeted analysis methods. Lastly, the obtained data may serve in the future as a basis for deciphering the biochemistry of truffles more deeply as well, when protein databases of the different truffle species will be more complete.

Brain-Derived Extracellular Vesicles in Health and Disease: A Methodological Perspective.

Extracellular vesicles (EVs) are double membrane structures released by presumably all cell types that transport and deliver lipids, proteins, and genetic material to near or distant recipient cells, thereby affecting their phenotype. The basic knowledge of their functions in healthy and diseased brain is still murky and many questions about their biology are unsolved. In neurological diseases, EVs are regarded as attractive biomarkers and as therapeutic tools due to their ability to cross the blood-brain barrier (BBB). EVs have been successfully isolated from conditioned media of primary brain cells and cerebrospinal fluid (CSF), but protocols allowing for the direct study of pathophysiological events mediated or influenced by EVs isolated from brain have only recently been published. This review aims to give a brief overview of the current knowledge of EVs' functions in the central nervous system (CNS) and the current protocols to isolate brain-derived EVs (BDEVs) used in different publications. By comparing the proteomic analysis of some of these publications, we also assess the influence of the isolation method on the protein content of BDEVs.

Proteogenomic analysis of Inhibitor of Differentiation 4 (ID4) in basal-like breast cancer.

BACKGROUND: Basal-like breast cancer (BLBC) is a poorly characterised, heterogeneous disease. Patients are diagnosed with aggressive, high-grade tumours and often relapse with chemotherapy resistance. Detailed understanding of the molecular underpinnings of this disease is essential to the development of personalised therapeutic strategies. Inhibitor of differentiation 4 (ID4) is a helix-loop-helix transcriptional regulator required for mammary gland development. ID4 is overexpressed in a subset of BLBC patients, associating with a stem-like poor prognosis phenotype, and is necessary for the growth of cell line models of BLBC through unknown mechanisms. METHODS: Here, we have defined unique molecular insights into the function of ID4 in BLBC and the related disease high-grade serous ovarian cancer (HGSOC), by combining RIME proteomic analysis, ChIP-seq mapping of genomic binding sites and RNA-seq. RESULTS: These studies reveal novel interactions with DNA damage response proteins, in particular, mediator of DNA damage checkpoint protein 1 (MDC1). Through MDC1, ID4 interacts with other DNA repair proteins (γH2AX and BRCA1) at fragile chromatin sites. ID4 does not affect transcription at these sites, instead binding to chromatin following DNA damage. Analysis of clinical samples demonstrates that ID4 is amplified and overexpressed at a higher frequency in BRCA1-mutant BLBC compared with sporadic BLBC, providing genetic evidence for an interaction between ID4 and DNA damage repair deficiency. CONCLUSIONS: These data link the interactions of ID4 with MDC1 to DNA damage repair in the aetiology of BLBC and HGSOC.

Inspired by Sea Urchins: Warburg Effect Mediated Selectivity of Novel Synthetic Non-Glycoside 1,4-Naphthoquinone-6S-Glucose Conjugates in Prostate Cancer.

The phenomenon of high sugar consumption by tumor cells is known as Warburg effect. It results from a high glycolysis rate, used by tumors as preferred metabolic pathway even in aerobic conditions. Targeting the Warburg effect to specifically deliver sugar conjugated cytotoxic compounds into tumor cells is a promising approach to create new selective drugs. We designed, synthesized, and analyzed a library of novel 6-S-(1,4-naphthoquinone-2-yl)-d-glucose chimera molecules (SABs)-novel sugar conjugates of 1,4-naphthoquinone analogs of the sea urchin pigments spinochromes, which have previously shown anticancer properties. A sulfur linker (thioether bond) was used to prevent potential hydrolysis by human glycoside-unspecific enzymes. The synthesized compounds exhibited a Warburg effect mediated selectivity to human prostate cancer cells (including highly drug-resistant cell lines). Mitochondria were identified as a primary cellular target of SABs. The mechanism of action included mitochondria membrane permeabilization, followed by ROS upregulation and release of cytotoxic mitochondrial proteins (AIF and cytochrome C) to the cytoplasm, which led to the consequent caspase-9 and -3 activation, PARP cleavage, and apoptosis-like cell death. These results enable us to further clinically develop these compounds for effective Warburg effect targeting.

Proteome profiling of Pseudomonas aeruginosa PAO1 identifies novel responders to copper stress.

BACKGROUND: The opportunistic pathogen, Pseudomonas aeruginosa is well known for its environmental and metabolic versatility, yet many of the functions of its gene-products remain to be fully elucidated. This study's objective was to illuminate the potential functions of under-described gene-products during the medically relevant copper-stress condition. RESULTS: We used data-independent acquisition mass spectrometry to quantitate protein expression changes associated with copper stress in P. aeruginosa PAO1. Approximately 2000 non-redundant proteins were quantified, with 78 proteins altering in abundance by +/- 1.5-fold or more when cultured to mid-log growth in the presence of 50 µM copper sulfate. One-third of those differentially expressed proteins have no prior established functional roles. CONCLUSIONS: This study provides evidence for the functional involvement of some specific proteins in enabling P. aeruginosa to survive under sub-lethal concentrations of copper. This further paves the way for targeted investigations into the specific mechanisms of their activity.

Proteomic phenotyping of metastatic melanoma reveals putative signatures of MEK inhibitor response and prognosis.

BACKGROUND: Genotyping of melanomas is used to identify patients for treatment with BRAF and MEK inhibitors, but clinical responses are highly variable. This study investigated the utility of protein expression phenotyping to provide an integrated assessment of gene expression programs in BRAF/NRAS melanoma which would be useful for prognosis and may predict response to MEK inhibition. METHODS: Mass spectrometry profiling of early passage cell lines established from Stage III cutaneous melanomas was conducted. Basal protein expression was correlated with in vitro response to the MEK inhibitor, selumetinib. Protein expression in a cohort of 32 drug naïve BRAF/NRAS metastatic melanoma specimens was examined. The prognostic utility of a subset of these proteins and mRNA transcripts from a separate cohort was determined. RESULTS: Unsupervised analysis of basal cell line protein abundances delineated response to selumetinib, but BRAF/NRAS genotype did not. Resistance was associated with functions including cell motility, cell adhesion and cytoskeletal organization. Several of these response biomarkers were observed in lymph node biospecimens and correlated with melanoma-specific survival. Loss of ICAM-1 protein and mRNA expression was a strong prognosticator of diminished survival in BRAF/NRAS mutant melanoma. CONCLUSIONS: These results demonstrate the utility of proteomic phenotyping to identify both putative biomarkers of response to MEK inhibition and prognostication associated with metastatic melanoma.

Application of Displacement Chromatography to Online Two-Dimensional Liquid Chromatography Coupled to Tandem Mass Spectrometry Improves Peptide Separation Efficiency and Detectability for the Analysis of Complex Proteomes.

The complexity of mammalian proteomes is a challenge in bottom-up proteomics. For a comprehensive proteome analysis, multidimensional separation strategies are necessary. Online two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS) combining strong cation exchange (SCX) in the first dimension with reversed-phase (RP) chromatography in the second dimension provides a powerful approach to analyze complex proteomes. Although the combination of SCX with RP chromatography provides a good orthogonality, only a moderate separation is achieved in the first dimension for peptides with two (+2) or three (+3) positive charges. The aim of this study was to improve the performance of online SCX-RP-MS/MS by applying displacement chromatography to the first separation dimension. Compared to gradient chromatography mode (GCM), displacement chromatography mode (DCM) was expected to improve the separation of +2-peptides and +3-peptides, thus reducing complexity and increasing ionization and detectability. The results show that DCM provided a separation of +2-peptides and +3-peptides in remarkably sharp zones with a low degree of coelution, thus providing fractions with significantly higher purities compared to GCM. In particular, +2-peptides were separated over several fractions, which was not possible to achieve in GCM. The better separation in DCM resulted in a higher reproducibility and significantly higher identification rates for both peptides and proteins including a 2.6-fold increase for +2-peptides. The higher number of identified peptides in DCM resulted in significantly higher protein sequence coverages and a considerably higher number of unique peptides per protein. Compared to conventionally used salt-based GCM, DCM increased the performance of online SCX-RP-MS/MS and enabled comprehensive proteome profiling in the low microgram range.

Multi-laboratory analysis of the variability of shipped samples for proteomics following non-cooled international transport.

Transporting biological samples such as cells or tissues is complicated by the need to maintain integrity and minimise modification and degradation, but this is economically costly as the samples must be shipped in a frozen state. This multi-laboratory study investigated sample variability introduced by non-cooled transport of dried peptide samples for proteomic analysis using mass spectrometry. Human cancer cell tryptic lysates were proteolysed and dried in Australia and shipped by air to Europe and China. Samples were measured using label free mass spectrometry on similar LC-MS systems at all three sites. Preparation and analysis of the specimens in this manner resulted in only minor differences in protein identification and showed high quantitative reproducibility amongst the participating laboratories. We examined any impact on peptide chemical modification and report no discrepancies compared to the starting, non-shipped sample. We conclude that transport of non-cooled, dried peptides has negligible effect on sample integrity for downstream LC-MS analysis and therefore represents a cost-effective option to facilitate international proteomic collaborations. Data is available via ProteomeXchange with identifier PXD008160.

SWATH Mass Spectrometry Performance Using Extended Peptide MS/MS Assay Libraries.

The use of data-independent acquisition methods such as SWATH for mass spectrometry based proteomics is usually performed with peptide MS/MS assay libraries which enable identification and quantitation of peptide peak areas. Reference assay libraries can be generated locally through information dependent acquisition, or obtained from community data repositories for commonly studied organisms. However, there have been no studies performed to systematically evaluate how locally generated or repository-based assay libraries affect SWATH performance for proteomic studies. To undertake this analysis, we developed a software workflow, SwathXtend, which generates extended peptide assay libraries by integration with a local seed library and delivers statistical analysis of SWATH-quantitative comparisons. We designed test samples using peptides from a yeast extract spiked into peptides from human K562 cell lysates at three different ratios to simulate protein abundance change comparisons. SWATH-MS performance was assessed using local and external assay libraries of varying complexities and proteome compositions. These experiments demonstrated that local seed libraries integrated with external assay libraries achieve better performance than local assay libraries alone, in terms of the number of identified peptides and proteins and the specificity to detect differentially abundant proteins. Our findings show that the performance of extended assay libraries is influenced by the MS/MS feature similarity of the seed and external libraries, while statistical analysis using multiple testing corrections increases the statistical rigor needed when searching against large extended assay libraries.

Improving Protein Detection Confidence Using SWATH-Mass Spectrometry with Large Peptide Reference Libraries.

Protein quantification using data-independent acquisition methods such as SWATH-MS most commonly relies on spectral matching to a reference MS/MS assay library. To enable deep proteome coverage and efficient use of existing data, in silico approaches have been described to use archived or publicly available large reference spectral libraries for spectral matching. Since implicit in the use of larger libraries is the increasing likelihood of false-discoveries, new workflows are needed to ensure high confidence in protein matching under these conditions. We present a workflow which introduces a range of filters and thresholds aimed at increasing confidence that the resulting proteins are reliably detected and their quantitation is consistent and reproducible. We demonstrated the workflow using extended libraries with SWATH data from human plasma samples and yeast-spiked human K562 cell lysate digest.