Omicron extensively but incompletely escapes Pfizer BNT162b2 neutralization.

The emergence of the SARS-CoV-2 variant of concern Omicron (Pango lineage B.1.1.529), first identified in Botswana and South Africa, may compromise vaccine effectiveness and lead to re-infections1. Here we investigated Omicron escape from neutralization by antibodies from South African individuals vaccinated with Pfizer BNT162b2. We used blood samples taken soon after vaccination from individuals who were vaccinated and previously infected with SARS-CoV-2 or vaccinated with no evidence of previous infection. We isolated and sequence-confirmed live Omicron virus from an infected person and observed that Omicron requires the angiotensin-converting enzyme 2 (ACE2) receptor to infect cells. We compared plasma neutralization of Omicron relative to an ancestral SARS-CoV-2 strain and found that neutralization of ancestral virus was much higher in infected and vaccinated individuals compared with the vaccinated-only participants. However, both groups showed a 22-fold reduction in vaccine-elicited neutralization by the Omicron variant. Participants who were vaccinated and had previously been infected exhibited residual neutralization of Omicron similar to the level of neutralization of the ancestral virus observed in the vaccination-only group. These data support the notion that reasonable protection against Omicron may be maintained using vaccination approaches.

MetaNovo: An open-source pipeline for probabilistic peptide discovery in complex metaproteomic datasets.

BACKGROUND: Microbiome research is providing important new insights into the metabolic interactions of complex microbial ecosystems involved in fields as diverse as the pathogenesis of human diseases, agriculture and climate change. Poor correlations typically observed between RNA and protein expression datasets make it hard to accurately infer microbial protein synthesis from metagenomic data. Additionally, mass spectrometry-based metaproteomic analyses typically rely on focused search sequence databases based on prior knowledge for protein identification that may not represent all the proteins present in a set of samples. Metagenomic 16S rRNA sequencing only targets the bacterial component, while whole genome sequencing is at best an indirect measure of expressed proteomes. Here we describe a novel approach, MetaNovo, that combines existing open-source software tools to perform scalable de novo sequence tag matching with a novel algorithm for probabilistic optimization of the entire UniProt knowledgebase to create tailored sequence databases for target-decoy searches directly at the proteome level, enabling metaproteomic analyses without prior expectation of sample composition or metagenomic data generation and compatible with standard downstream analysis pipelines. RESULTS: We compared MetaNovo to published results from the MetaPro-IQ pipeline on 8 human mucosal-luminal interface samples, with comparable numbers of peptide and protein identifications, many shared peptide sequences and a similar bacterial taxonomic distribution compared to that found using a matched metagenome sequence database-but simultaneously identified many more non-bacterial peptides than the previous approaches. MetaNovo was also benchmarked on samples of known microbial composition against matched metagenomic and whole genomic sequence database workflows, yielding many more MS/MS identifications for the expected taxa, with improved taxonomic representation, while also highlighting previously described genome sequencing quality concerns for one of the organisms, and identifying an experimental sample contaminant without prior expectation. CONCLUSIONS: By estimating taxonomic and peptide level information directly on microbiome samples from tandem mass spectrometry data, MetaNovo enables the simultaneous identification of peptides from all domains of life in metaproteome samples, bypassing the need for curated sequence databases to search. We show that the MetaNovo approach to mass spectrometry metaproteomics is more accurate than current gold standard approaches of tailored or matched genomic sequence database searches, can identify sample contaminants without prior expectation and yields insights into previously unidentified metaproteomic signals, building on the potential for complex mass spectrometry metaproteomic data to speak for itself.

A review of mass spectrometry-based analyses to understand COVID-19 convalescent plasma mechanisms of action.

The spread of coronavirus disease 2019 (COVID-19) viral pneumonia caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a worldwide pandemic claiming several thousands of lives worldwide. During this pandemic, several studies reported the use of COVID-19 convalescent plasma (CCP) from recovered patients to treat severely or critically ill patients. Although this historical and empirical treatment holds immense potential as a first line of response against eventual future unforeseen viral epidemics, there are several concerns regarding the efficacy and safety of this approach. This critical review aims to pinpoint the possible role of mass spectrometry-based analysis in the identification of unique molecular component proteins, peptides, and metabolites of CCP that explains the therapeutic mechanism of action against COVID-19. Additionally, the text critically reviews the potential application of mass spectrometry approaches in the search for novel plasma biomarkers that may enable a rapid and accurate assessment of the safety and efficacy of CCP. Considering the relative low-cost value involved in the CCP therapy, this proposed line of research represents a tangible scientific challenge that will be translated into clinical practice and help save several thousand lives around the world, specifically in low- and middle-income countries.

Immunogenicity of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infection and Ad26.CoV2.S Vaccination in People Living With Human Immunodeficiency Virus (HIV).

BACKGROUND: People living with HIV (PLWH) have been reported to have a higher risk of more severe COVID-19 disease and death. We assessed the ability of the Ad26.CoV2.S vaccine to elicit neutralizing activity against the Delta variant in PLWH relative to HIV-negative individuals. We also examined effects of HIV status and suppression on Delta neutralization response in SARS-CoV-2-infected unvaccinated participants. METHODS: We enrolled participants who were vaccinated through the SISONKE South African clinical trial of the Ad26.CoV2.S vaccine in healthcare workers (HCWs). PLWH in this group had well-controlled HIV infection. We also enrolled unvaccinated participants previously infected with SARS-CoV-2. Neutralization capacity was assessed by a live virus neutralization assay of the Delta variant. RESULTS: Most Ad26.CoV2.S vaccinated HCWs were previously infected with SARS-CoV-2. In this group, Delta variant neutralization was 9-fold higher compared with the infected-only group and 26-fold higher relative to the vaccinated-only group. No decrease in Delta variant neutralization was observed in PLWH relative to HIV-negative participants. In contrast, SARS-CoV-2-infected, unvaccinated PLWH showed 7-fold lower neutralization and a higher frequency of nonresponders, with the highest frequency of nonresponders in people with HIV viremia. Vaccinated-only participants showed low neutralization capacity. CONCLUSIONS: The neutralization response of the Delta variant following Ad26.CoV2.S vaccination in PLWH with well-controlled HIV was not inferior to HIV-negative participants, irrespective of past SARS-CoV-2 infection. In SARS-CoV-2-infected and nonvaccinated participants, HIV infection reduced the neutralization response to SARS-CoV-2, with the strongest reduction in HIV viremic individuals.

Nuclear transport proteins are secreted by cancer cells and identified as potential novel cancer biomarkers.

Previous studies have identified increased expression of members of the nuclear transport protein family in cancer cells. Recently, certain nuclear transport proteins have been reported to be secreted by cells and found in the serum. The aims of our study were to investigate the levels of multiple nuclear transport proteins secreted from cancer cells, and to determine their potential as diagnostic markers for cervical and oesophageal cancer. Mass spectrometry identified 10 nuclear transport proteins in the secretome and exosomes of cultured cancer cells, and Western blot analysis confirmed increased secreted levels in cancer cells compared to normal. To investigate their presence in patient serum, enzyme-linked immunosorbent assays were performed and revealed significantly increased levels of KPNß1, CRM1, CAS, IPO5 and TNPO1 in cervical and oesophageal cancer patient serum compared to non-cancer controls. Significantly elevated KPNα2 and RAN levels were also identified in oesophageal cancer serum samples. Logistics regression analyses revealed IPO5 and TNPO1 to be the best performing individual candidate biomarkers in discriminating between cancer cases and controls. The combination of KPNß1, CRM1, KPNα2, CAS, RAN, IPO5 and TNPO1 as a panel of biomarkers had the highest diagnostic capacity with an area under the curve of 0.944 and 0.963, for cervical cancer and oesophageal cancer, and sensitivity of 92.5% at 86.8% specificity and 95.3% sensitivity at 87.5% specificity, respectively. These results suggest that nuclear transport proteins have potential as diagnostic biomarkers for cervical and oesophageal cancers, with a combination of protein family members being the best predictor.

A highly predictive autoantibody-based biomarker panel for prognosis in early-stage NSCLC with potential therapeutic implications.

BACKGROUND: Lung cancer is the leading cause of cancer-related death worldwide. Surgical resection remains the definitive curative treatment for early-stage disease offering an overall 5-year survival rate of 62%. Despite careful case selection, a significant proportion of early-stage cancers relapse aggressively within the first year post-operatively. Identification of these patients is key to accurate prognostication and understanding the biology that drives early relapse might open up potential novel adjuvant therapies. METHODS: We performed an unsupervised interrogation of >1600 serum-based autoantibody biomarkers using an iterative machine-learning algorithm. RESULTS: We identified a 13 biomarker signature that was highly predictive for survivorship in post-operative early-stage lung cancer; this outperforms currently used autoantibody biomarkers in solid cancers. Our results demonstrate significantly poor survivorship in high expressers of this biomarker signature with an overall 5-year survival rate of 7.6%. CONCLUSIONS: We anticipate that the data will lead to the development of an off-the-shelf prognostic panel and further that the oncogenic relevance of the proteins recognised in the panel may be a starting point for a new adjuvant therapy.

Mechanistic implications of altered protein expression in rheumatic heart disease.

Rheumatic heart disease (RHD) is a major cause of cardiovascular morbidity and mortality in low- and middle-income countries, where living conditions promote spread of group A ß-haemolytic streptococcus. Autoimmune reactions due to molecular mimicry of bacterial epitopes by host proteins cause acute rheumatic fever (ARF) and subsequent disease progression to RHD. Despite knowledge of the factors that predispose to ARF and RHD, determinants of the progression to valvular damage and the molecular events involved remain incompletely characterised. This review focuses on altered protein expression in heart valves, myocardial tissue and plasma of patients with RHD and pathogenic consequences on RHD. Proteins mainly involved in structural organization of the valve matrix, blood homeostasis and immune response were altered due to RHD pathogenesis. Study of secreted forms of these proteins may aid the development of non-invasive biomarkers for early diagnosis and monitoring outcomes in RHD. Valve replacement surgery, the single evidence-based strategy to improve outcomes in severe RHD, is costly, largely unavailable in low- and middle-income countries (LMIC) and requires specialised facilities. When diagnosed early, penicillin prophylaxis may be used to delay progression to severe valvular damage. Echocardiography and cardiovascular magnetic resonance and the standard imaging tools recommended to confirm early diagnosis remain largely unavailable and inaccessible in most LMIC and both require expensive equipment and highly skilled persons for manipulation as well as interpretation of results. Changes in protein expression in heart valves and myocardium are associated with progressive valvular deformation in RHD. Understanding these protein changes should shed more light on the mechanisms of pathogenicity, while secreted forms of these proteins may provide leads towards a biomarker for non-invasive early detection of RHD.

Phosphoproteomic Approaches to Discover Novel Substrates of Mycobacterial Ser/Thr Protein Kinases.

Mycobacterial Ser/Thr protein kinases (STPKs) play a critical role in signal transduction pathways that ultimately determine mycobacterial growth and metabolic adaptation. Identification of key physiological substrates of these protein kinases is, therefore, crucial to better understand how Ser/Thr phosphorylation contributes to mycobacterial environmental adaptation, including response to stress, cell division, and host-pathogen interactions. Various substrate detection methods have been employed with limited success, with direct targets of STPKs remaining elusive. Recently developed mass spectrometry (MS)-based phosphoproteomic approaches have expanded the list of potential STPK substrate identifications, yet further investigation is required to define the most functionally significant phosphosites and their physiological importance. Prior to the application of MS workflows, for instance, GarA was the only known and validated physiological substrate for protein kinase G (PknG) from pathogenic mycobacteria. A subsequent list of at least 28 candidate PknG substrates has since been reported with the use of MS-based analyses. Herein, we integrate and critically review MS-generated datasets available on novel STPK substrates and report new functional and subcellular localization enrichment analyses on novel candidate protein kinase A (PknA), protein kinase B (PknB) and PknG substrates to deduce the possible physiological roles of these kinases. In addition, we assess substrate specificity patterns across different mycobacterial STPKs by analyzing reported sets of phosphopeptides, in order to determine whether novel motifs or consensus regions exist for mycobacterial Ser/Thr phosphorylation sites. This review focuses on MS-based techniques employed for STPK substrate identification in mycobacteria, while highlighting the advantages and challenges of the various applications.

Cell Envelope Proteomics of Mycobacteria.

The World Health Organization (WHO) estimates that Mycobacterium tuberculosis, the most pathogenic mycobacterium species to humans, has infected up to a quarter of the world's population, with the occurrence of multidrug-resistant strains on the rise. Research into the detailed composition of the cell envelope proteome in mycobacteria over the last 20 years has formed a key part of the efforts to understand host-pathogen interactions and to control the current tuberculosis epidemic. This is due to the great importance of the cell envelope proteome during infection and during the development of antibiotic resistance as well as the search of surface-exposed proteins that could be targeted by therapeutics and vaccines. A variety of experimental approaches and mycobacterial species have been used in proteomic studies thus far. Here we provide for the first time an extensive summary of the different approaches to isolate the mycobacterial cell envelope, highlight some of the limitations of the studies performed thus far, and comment on how the recent advances in membrane proteomics in other fields might be translated into the field of mycobacteria to provide deeper coverage.

The cyanobacterial lectin, microvirin-N, enhances the specificity and sensitivity of lipoarabinomannan-based TB diagnostic tests.

Tuberculosis (TB) is one of the top ten causes of death globally, despite being treatable. The eradication of TB disease requires, amongst others, diagnostic tests with high specificity and sensitivity that will work at the point of care (POC) in low-resource settings. The TB surface glycolipid antigen, mannose-capped lipoarabinomannan (ManLAM) currently serves as the only POC molecular diagnostic biomarker suitable for use in low cost immunoassays. Here, we demonstrate the high affinity and exceptional specificity of microvirin-N (MVN), a 14.3 kDa cyanobacterial lectin, toward H37Rv TB ManLAM and utilize it to develop a novel on-bead ELISA. MVN binds to ManLAM with sub-picomolar binding affinity, but does not bind to other variants of LAM expressed by non-pathogenic mycobacteria - a level of binding specificity and affinity that current commercially available anti-LAM antibodies cannot achieve. An on-bead ELISA was subsequently developed using MVN-functionalized magnetic beads which allows for the specific capture of ManLAM from human urine with a limit of detection (LOD) of 1.14 ng mL-1 and no cross-reactivity when tested with PILAM, a variant of LAM found on non-pathogenic mycobacteria.

Identification of Novel Physiological Substrates of Mycobacterium bovis BCG Protein Kinase G (PknG) by Label-free Quantitative Phosphoproteomics.

Mycobacterial Ser/Thr kinases play a critical role in bacterial physiology and pathogenesis. Linking kinases to the substrates they phosphorylate in vivo, thereby elucidating their exact functions, is still a challenge. The aim of this work was to associate protein phosphorylation in mycobacteria with important subsequent macro cellular events by identifying the physiological substrates of PknG in Mycobacterium bovis BCG. The study compared the phosphoproteome dynamics during the batch growth of M. bovis BCG versus the respective PknG knock-out mutant (ΔPknG-BCG) strains. We employed TiO2 phosphopeptide enrichment techniques combined with label-free quantitative phosphoproteomics workflow on LC-MS/MS. The comprehensive analysis of label-free data identified 603 phosphopeptides on 307 phosphoproteins with high confidence. Fifty-five phosphopeptides were differentially phosphorylated, of these, 23 phosphopeptides were phosphorylated in M. bovis BCG wild-type only and not in the mutant. These were further validated through targeted mass spectrometry assays (PRMs). Kinase-peptide docking studies based on a published crystal structure of PknG in complex with GarA revealed that the majority of identified phosphosites presented docking scores close to that seen in previously described PknG substrates, GarA, and ribosomal protein L13. Six out of the 22 phosphoproteins had higher docking scores than GarA, consistent with the proteins identified here being true PknG substrates. Based on protein functional analysis of the PknG substrates identified, this study confirms that PknG plays an important regulatory role in mycobacterial metabolism, through phosphorylation of ATP binding proteins and enzymes in the TCA cycle. This work also reinforces PknG's regulation of protein translation and folding machinery.

B cells and antibody production in melanoma.

Recent developments in the immuno-oncology field strongly support a role for the immune system in both the prevention and progression of melanoma. Melanoma is a highly immunogenic cancer, including its ability to induce tumour antigen-specific B cell and antibody responses through largely unknown mechanisms. This review considers likely hypothetical mechanisms by which anti-tumour surveillance detects pre-cancerous cells and by which immune (including B cell and antibody) responses may be elicited during malignancy. The review further considers potential pro- and anti-tumour functions of B cells and antibodies (including tertiary lymphoid structures) in both the tumour microenvironment and in circulation. Although the vast majority of studies have focused on T cells, recent evidence highlights the important roles of B cells in response to malignancy. B cells and antibodies are also discussed in the context of their potential utility as clinical biomarkers for various applications (as diagnostic, prognostic, therapeutic efficacy, and toxicity proxies), with a particular focus on protein microarray-based antibody detection and quantitation. Although the role of B cells in melanoma is incompletely understood, the measurement of circulating tumour-specific antibodies represents a promising avenue in the search for melanoma-relevant biomarkers.

The Role of Tumor Microenvironment in Chemoresistance: 3D Extracellular Matrices as Accomplices.

BACKGROUND: The functional interplay between tumor cells and their adjacent stroma has been suggested to play crucial roles in the initiation and progression of tumors and the effectiveness of chemotherapy. The extracellular matrix (ECM), a complex network of extracellular proteins, provides both physical and chemicals cues necessary for cell proliferation, survival, and migration. Understanding how ECM composition and biomechanical properties affect cancer progression and response to chemotherapeutic drugs is vital to the development of targeted treatments. METHODS: 3D cell-derived-ECMs and esophageal cancer cell lines were used as a model to investigate the effect of ECM proteins on esophageal cancer cell lines response to chemotherapeutics. Immunohistochemical and qRT-PCR evaluation of ECM proteins and integrin gene expression was done on clinical esophageal squamous cell carcinoma biopsies. Esophageal cancer cell lines (WHCO1, WHCO5, WHCO6, KYSE180, KYSE 450 and KYSE 520) were cultured on decellularised ECMs (fibroblasts-derived ECM; cancer cell-derived ECM; combinatorial-ECM) and treated with 0.1% Dimethyl sulfoxide (DMSO), 4.2 µM cisplatin, 3.5 µM 5-fluorouracil and 2.5 µM epirubicin for 24 h. Cell proliferation, cell cycle progression, colony formation, apoptosis, migration and activation of signaling pathways were used as our study endpoints. RESULTS: The expression of collagens, fibronectin and laminins was significantly increased in esophageal squamous cell carcinomas (ESCC) tumor samples compared to the corresponding normal tissue. Decellularised ECMs abrogated the effect of drugs on cancer cell cycling, proliferation and reduced drug induced apoptosis by 20⁻60% that of those plated on plastic. The mitogen-activated protein kinase-extracellular signal-regulated kinase (MEK-ERK) and phosphoinositide 3-kinase-protein kinase B (PI3K/Akt) signaling pathways were upregulated in the presence of the ECMs. Furthermore, our data show that concomitant addition of chemotherapeutic drugs and the use of collagen- and fibronectin-deficient ECMs through siRNA inhibition synergistically increased cancer cell sensitivity to drugs by 30⁻50%, and reduced colony formation and cancer cell migration. CONCLUSION: Our study shows that ECM proteins play a key role in the response of cancer cells to chemotherapy and suggest that targeting ECM proteins can be an effective therapeutic strategy against chemoresistant tumors.

Quantitative proteomic analysis of HIV-1 Tat-induced dysregulation in SH-SY5Y neuroblastoma cells.

Despite affecting up to 70% of HIV-positive patients and being the leading cause of dementia in patients under 40 years, the molecular mechanisms involved in the onset of HIV-associated neurocognitive disorders (HAND) are not well understood. To address this, we performed SILAC-based quantitative proteomic analysis on HIV-Tat treated SH-SY5Y neuroblastoma cells. Isolated protein was fractionated by SDS-PAGE and analyzed by nLC-MS/MS on an Orbitrap Velos. Using MaxQuant, we identified and quantified 3077 unique protein groups, of which 407 were differentially regulated. After applying an additional standard deviation-based cutoff, 29 of these were identified as highly significantly and stably dysregulated. GO term analysis shows dysregulation in both protein translation machinery as well as cytoskeletal regulation that have both been implicated in other dementias. In addition, several key cytoskeletal regulatory proteins such as ARHGEF17, the Rho GTPase, SHROOM3, and CMRP1 are downregulated. Together, these data demonstrate that HIV-Tat can dysregulate neuronal cytoskeletal regulatory proteins that could lead to the major HAND clinical manifestation-synapse loss.

Advances in the development of human protein microarrays.

INTRODUCTION: High-content protein microarrays in principle enable the functional interrogation of the human proteome in a broad range of applications, including biomarker discovery, profiling of immune responses, identification of enzyme substrates, and quantifying protein-small molecule, protein-protein and protein-DNA/RNA interactions. As with other microarrays, the underlying proteomic platforms are under active technological development and a range of different protein microarrays are now commercially available. However, deciphering the differences between these platforms to identify the most suitable protein microarray for the specific research question is not always straightforward. Areas covered: This review provides an overview of the technological basis, applications and limitations of some of the most commonly used full-length, recombinant protein and protein fragment microarray platforms, including ProtoArray Human Protein Microarrays, HuProt Human Proteome Microarrays, Human Protein Atlas Protein Fragment Arrays, Nucleic Acid Programmable Arrays and Immunome Protein Arrays. Expert commentary: The choice of appropriate protein microarray platform depends on the specific biological application in hand, with both more focused, lower density and higher density arrays having distinct advantages. Full-length protein arrays offer advantages in biomarker discovery profiling applications, although care is required in ensuring that the protein production and array fabrication methodology is compatible with the required downstream functionality.

TB-IRIS: Proteomic analysis of in vitro PBMC responses to Mycobacterium tuberculosis and response modulation by dexamethasone.

Paradoxical tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) occurs in 8-54% of South African patients undergoing treatment for tuberculosis/human immunodeficiency virus co-infection. Improved TB-IRIS molecular pathogenesis understanding would enhance risk stratification, diagnosis, prognostication, and treatment. We assessed how TB-IRIS status and dexamethasone influence leukocyte proteomic responses to Mycobacterium tuberculosis (Mtb). Patient blood was obtained three weeks post-anti-retroviral therapy initiation. Isolated mononuclear cells were stimulated ex vivo with heat-killed Mtb in the presence/absence of dexamethasone. Mass spectrometry-based proteomic comparison of TB-IRIS and non-IRIS patient-derived cells facilitated generation of hypotheses regarding pathogenesis. Few represented TB-IRIS-group immune-related pathways achieved significant activation, with relative under-utilisation of "inter-cellular interaction" and "Fcγ receptor-mediated phagocytosis" (but a tendency towards apoptosis-related) pathways. Dexamethasone facilitated significant activation of innate-related pathways. Differentially-expressed non-IRIS-group proteins suggest focused and co-ordinated immunological pathways, regardless of dexamethasone status. Findings suggest a relative deficit in TB-IRIS-group responses to and clearance of Mtb antigens, ameliorated by dexamethasone.

In silico verification and parallel reaction monitoring prevalidation of potential prostate cancer biomarkers.

PURPOSE: Targeted proteomics of potential biomarkers is often challenging. Hence, we developed an intermediate workflow to streamline potential urinary biomarkers of prostate cancer (PCa). MATERIALS & METHODS: Using previously discovered potential PCa biomarkers; we selected proteotypic peptides for targeted validation. Preliminary in silico immunohistochemical and single reaction monitoring (SRM) verification was performed. Successful PTPs were then prevalidated using parallel reaction monitoring (PRM) and reconfirmed in 15 publicly available databases. RESULTS: Stringency-based targetable potential biomarkers were shortlisted following in silico screening. PRM reveals top 12 potential biomarkers including the top ranking seven in silico verification-based biomarkers. Database reconfirmation showed differential expression between PCa and benign/normal prostatic urine samples. CONCLUSION: The pragmatic penultimate screening step, described herein, would immensely improve targeted proteomics validation of potential disease biomarkers.

Comparative reevaluation of FASP and enhanced FASP methods by LC-MS/MS.

Filter-aided sample preparation is a proteomic technique for the preparation and on column proteolysis of proteins. Recently an enhanced FASP protocol was developed that uses deoxycholic acid (DCA) and that reportedly enhances trypsin proteolysis, resulting in increases cytosolic and membrane protein representation. FASP and eFASP were re-evaluated by ultra-high-performance liquid chromatography coupled to a quadrupole mass filter Orbitrap analyzer (Q Exactive). Although there was no difference in trypsin activity, 14,099 and 13,414 peptides, describing 1723 and 1793 protein groups, from Escherichia coli K12 were identified using FASP and eFASP, respectively. Characterization of the physicochemical properties of identified peptides showed no significant differences other than eFASP extracting slightly more basic peptides. At the protein level, both methods extracted essentially the same number of hydrophobic transmembrane helix-containing proteins as well as proteins associated with the cytoplasm or the cytoplasmic and outer membranes. By employing state-of-the-art LC-MS/MS shot gun proteomics, our results indicate that FASP and eFASP showed no significant differences at the protein level. However, because of the slight differences in selectivity at the physicochemical level of peptides, these methods can be seen to be somewhat complementary for analyses of complex peptide mixtures.

Mycobacterial proteomics: analysis of expressed proteomes and post-translational modifications to identify candidate virulence factors.

The Mycobacterium tuberculosis bacillus has a number of unique features that make it a particularly effective human pathogen. Although genomic analysis has added to our current understanding of the molecular basis by which M. tuberculosis damages its host, proteomics may be better suited to describe the dynamic interactions between mycobacterial and host systems that underpin this disease. The M. tuberculosis proteome has been investigated using proteomics for over a decade, with increasingly sophisticated mass spectrometry technology and sensitive methods for comparative proteomic profiling. Deeper coverage of the M. tuberculosis proteome has led to the identification of hundreds of putative virulence determinants, as well as an unsurpassed coverage of post-translational modifications. Proteomics is therefore uniquely poised to contribute to our understanding of this pathogen, which may ultimately lead to better management of the disease.

Humoral immunoprofiling identifies novel biomarkers and an immune suppressive autoantibody phenotype at the site of disease in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a heterogeneous cancer, with minimal response to therapeutic intervention and with 85% of cases diagnosed at an advanced stage due to lack of early symptoms, highlighting the importance of understanding PDAC immunology in greater detail. Here, we applied an immunoproteomic approach to investigate autoantibody responses against cancer-testis and tumor-associated antigens in PDAC using a high-throughput multiplexed protein microarray platform, comparing humoral immune responses in serum and at the site of disease in order to shed new light on immune responses in the tumor microenvironment. We simultaneously quantified serum or tissue IgG and IgA antibody isotypes and subclasses in a cohort of PDAC, disease control and healthy patients, observing inter alia that subclass utilization in tumor tissue samples was predominantly immune suppressive IgG4 and inflammatory IgA2, contrasting with predominant IgG3 and IgA1 subclass utilization in matched sera and implying local autoantibody production at the site of disease in an immune-tolerant environment. By comparison, serum autoantibody subclass profiling for the disease controls identified IgG4, IgG1, and IgA1 as the abundant subclasses. Combinatorial analysis of serum autoantibody responses identified panels of candidate biomarkers. The top IgG panel included ACVR2B, GAGE1, LEMD1, MAGEB1 and PAGE1 (sensitivity, specificity and AUC values of 0.933, 0.767 and 0.906). Conversely, the top IgA panel included AURKA, GAGE1, MAGEA10, PLEKHA5 and XAGE3aV1 (sensitivity, specificity, and AUC values of 1.000, 0.800, and 0.954). Assessment of antigen-specific serum autoantibody glycoforms revealed abundant sialylation on IgA in PDAC, consistent with an immune suppressive IgA response to disease.