Deciphering Isomers with a Multiple Reaction Monitoring Method for the Complete Detectable O-Glycan Repertoire of the Candidate Therapeutic, Lubricin.

Glycosylation is a fundamental post-translational modification, occurring on half of all proteins. Despite its significance, our understanding is limited, in part due to the inherent difficulty in studying these branched, multi-isomer structures. Accessible, detailed, and quantifiable methods for studying glycans, particularly O-glycans, are needed. Here we take a multiple reaction monitoring (MRM) approach to differentiate and relatively quantify all detectable glycans, including isomers, on the heavily O-glycosylated protein lubricin. Lubricin (proteoglycan 4) is essential for lubrication of the joint and eye. Given the therapeutic potential of lubricin, it is essential to understand its O-glycan repertoire in biological and recombinantly produced samples. O-Glycans were released by reductive ß-elimination and defined, showing a range of 26 neutral, sulfated, sialylated, and both sulfated and sialylated core 1 (Galß1-3GalNAcα1-) and core 2 (Galß1-3(GlcNAcß1-6)GalNAcα1-) structures. Isomer-specific MRM transitions allowed effective differentiation of neutral glycan isomers as well as sulfated isomeric structures, where the sulfate was retained on the fragment ions. This strategy was not as effective with labile sialylated structures; instead, it was observed that the optimal collision energy for the m/z 290.1 sialic acid B-fragment differed consistently between sialic acid isomers, allowing differentiation between isomers when fragmentation spectra were insufficient. This approach was also effective for purchased Neu5Acα2-3Galß1-4Glc and Neu5Acα2-6Galß1-4Glc and for Neu5Acα2-3Galß1-4GlcNAc and Neu5Acα2-6Galß1-4GlcNAc linkage isomers with the Neu5Acα2-6 consistently requiring more energy for optimal generation of the m/z 290.1 fragment. Overall, this method provides an effective and easily accessible approach for the quantification and annotation of complex released O-glycan samples.

Separation of Sialylated Glycan Isomers by Differential Mobility Spectrometry.

Mass spectrometry has proven itself to be an important technology for characterizing intact glycoproteins, glycopeptides, and released glycans. However, these molecules often present significant challenges during analysis. For example, glycans of identical molecular weights can be present in many isomeric forms, with one form having dramatically more biological activity than the others. Discriminating among these isomeric forms using mass spectrometry alone can be daunting, which is why orthogonal techniques, such as ion mobility spectrometry, have been explored. Here, we demonstrate the use of differential mobility spectrometry (DMS) to separate isomeric glycans differing only in the linkages of sialic acid groups (e.g., α 2,3 versus α 2,6). This ability extends from a small trisaccharide species to larger biantennary systems and is driven, in part, by the role of intramolecular solvation of the charge site(s) on these ions within the DMS environment.

Assessment of SRM, MRM(3) , and DIA for the targeted analysis of phosphorylation dynamics in non-small cell lung cancer.

Hypothesis-driven MS-based targeted proteomics has gained great popularity in a relatively short timespan. Next to the widely established selected reaction monitoring (SRM) workflow, data-independent acquisition (DIA), also referred to as sequential window acquisition of all theoretical spectra (SWATH) was introduced as a high-throughput targeted proteomics method. DIA facilitates increased proteome coverage, however, does not yet reach the sensitivity obtained with SRM. Therefore, a well-informed method selection is crucial for designing a successful targeted proteomics experiment. This is especially the case when targeting less conventional peptides such as those that contain PTMs, as these peptides do not always adhere to the optimal fragmentation considerations for targeted assays. Here, we provide insight into the performance of DIA, SRM, and MRM cubed (MRM(3) ) in the analysis of phosphorylation dynamics throughout the phosphoinositide 3-kinase mechanistic target of rapamycin (PI3K-mTOR) and mitogen-activated protein kinase (MAPK) signaling network. We observe indeed that DIA is less sensitive when compared to SRM, however demonstrates increased flexibility, by postanalysis selection of alternative phosphopeptide precursors. Additionally, we demonstrate the added benefit of MRM(3) , allowing the quantification of two poorly accessible phosphosites. In total, targeted proteomics enabled the quantification of 42 PI3K-mTOR and MAPK phosphosites, gaining a so far unachieved in-depth view mTOR signaling events linked to tyrosine kinase inhibitor resistance in non-small cell lung cancer.

Comparing Multiple Reaction Monitoring and Sequential Window Acquisition of All Theoretical Mass Spectra for the Relative Quantification of Barley Gluten in Selectively Bred Barley Lines.

Celiac disease (CD) is a disease of the small intestine that occurs in genetically susceptible subjects triggered by the ingestion of cereal gluten proteins for which the only treatment is strict adherence to a life-long gluten-free diet. Barley contains four gluten protein families, and the existence of barley genotypes that do not accumulate the B-, C-, and D-hordeins paved the way for the development of an ultralow gluten phenotype. Using conventional breeding strategies, three null mutations behaving as recessive alleles were combined to create a hordein triple-null barley variety. Proteomics has become an invaluable tool for characterization and quantification of the protein complement of cereal grains. In this study multiple reaction monitoring (MRM) mass spectrometry, viewed as the gold standard for peptide quantification, was compared to the data-independent acquisition strategy known as SWATH-MS (sequential window acquisition of all theoretical mass spectra). SWATH-MS was comparable (p < 0.001) to MRM-MS for 32/33 peptides assessed across the four families of hordeins (gluten) in eight barley lines. The results of SWATH-MS analysis further confirmed the absence of the B-, C-, and D-hordeins in the triple-null barley line and showed significantly reduced levels ranging from <1% to 16% relative to wild-type (WT) cv Sloop for the minor γ-hordein class. SWATH-MS represents a valuable tool for quantitative proteomics based on its ability to generate reproducible data comparable with MRM-MS, but has the added benefits of allowing reinterrogation of data to improve analytical performance, ask new questions, and in this case perform quantification of trypsin-resistant proteins (C-hordeins) through analysis of their semi- or nontryptic fragments.

Selected reaction monitoring to differentiate and relatively quantitate isomers of sulfated and unsulfated core 1 O-glycans from salivary MUC7 protein in rheumatoid arthritis.

Rheumatoid arthritis is a common and debilitating systemic inflammatory condition affecting up to 1% of the world's population. This study aimed to investigate the immunological significance of O-glycans in chronic arthritis at a local and systemic level. O-Glycans released from synovial glycoproteins during acute and chronic arthritic conditions were compared and immune-reactive glycans identified. The sulfated core 1 O-glycan (Galß1-3GalNAcol) was immune reactive, showing a different isomeric profile in the two conditions. From acute reactive arthritis, three isomers could be sequenced, but in patients with chronic rheumatoid arthritis, only a single 3-Gal sulfate-linked isomer could be identified. The systemic significance of this glycan epitope was investigated using the salivary mucin MUC7 in patients with rheumatoid arthritis and normal controls. To analyze this low abundance glycan, a selected reaction monitoring (SRM) method was developed to differentiate and relatively quantitate the core 1 O-glycan and the sulfated core 1 O-glycan Gal- and GalNAc-linked isomers. The acquisition of highly sensitive full scan linear ion trap MS/MS spectra in addition to quantitative SRM data allowed the 3- and 6-linked Gal isomers to be differentiated. The method was used to relatively quantitate the core 1 glycans from MUC7 to identify any systemic changes in this carbohydrate epitope. A statistically significant increase in sulfation was identified in salivary MUC7 from rheumatoid arthritis patients. This suggests a potential role for this epitope in chronic inflammation. This study was able to develop an SRM approach to specifically identify and relatively quantitate sulfated core 1 isomers and the unsulfated structure. The expansion of this method may afford an avenue for the high throughput investigation of O-glycans.

A multiplex protein panel assay for severity prediction and outcome prognosis in patients with COVID-19: An observational multi-cohort study.

Background: Global healthcare systems continue to be challenged by the COVID-19 pandemic, and there is a need for clinical assays that can help optimise resource allocation, support treatment decisions, and accelerate the development and evaluation of new therapies. Methods: We developed a multiplexed proteomics assay for determining disease severity and prognosis in COVID-19. The assay quantifies up to 50 peptides, derived from 30 known and newly introduced COVID-19-related protein markers, in a single measurement using routine-lab compatible analytical flow rate liquid chromatography and multiple reaction monitoring (LC-MRM). We conducted two observational studies in patients with COVID-19 hospitalised at Charité - Universitätsmedizin Berlin, Germany before (from March 1 to 26, 2020, n=30) and after (from April 4 to November 19, 2020, n=164) dexamethasone became standard of care. The study is registered in the German and the WHO International Clinical Trials Registry (DRKS00021688). Findings: The assay produces reproducible (median inter-batch CV of 10.9%) absolute quantification of 47 peptides with high sensitivity (median LLOQ of 143 ng/ml) and accuracy (median 96.8%). In both studies, the assay reproducibly captured hallmarks of COVID-19 infection and severity, as it distinguished healthy individuals, mild, moderate, and severe COVID-19. In the post-dexamethasone cohort, the assay predicted survival with an accuracy of 0.83 (108/130), and death with an accuracy of 0.76 (26/34) in the median 2.5 weeks before the outcome, thereby outperforming compound clinical risk assessments such as SOFA, APACHE II, and ABCS scores. Interpretation: Disease severity and clinical outcomes of patients with COVID-19 can be stratified and predicted by the routine-applicable panel assay that combines known and novel COVID-19 biomarkers. The prognostic value of this assay should be prospectively assessed in larger patient cohorts for future support of clinical decisions, including evaluation of sample flow in routine setting. The possibility to objectively classify COVID-19 severity can be helpful for monitoring of novel therapies, especially in early clinical trials. Funding: This research was funded in part by the European Research Council (ERC) under grant agreement ERC-SyG-2020 951475 (to M.R) and by the Wellcome Trust (IA 200829/Z/16/Z to M.R.). The work was further supported by the Ministry of Education and Research (BMBF) as part of the National Research Node 'Mass Spectrometry in Systems Medicine (MSCoresys)', under grant agreements 031L0220 and 161L0221. J.H. was supported by a Swiss National Science Foundation (SNSF) Postdoc Mobility fellowship (project number 191052). This study was further supported by the BMBF grant NaFoUniMedCOVID-19 - NUM-NAPKON, FKZ: 01KX2021. The study was co-funded by the UK's innovation agency, Innovate UK, under project numbers 75594 and 56328.

Direct evidence for the formation of diastereoisomeric benzylpenicilloyl haptens from benzylpenicillin and benzylpenicillenic acid in patients.

Covalent binding to proteins to form neoantigens is thought to be central to the pathogenesis of penicillin hypersensitivity reactions. We have undertaken detailed mass spectrometric studies to define the mechanism and protein chemistry of hapten formation from benzylpenicillin (BP) and its rearrangement product, benzylpenicillenic acid (PA). Mass spectrometric analysis of human serum albumin exposed to BP and PA in vitro revealed that at low concentrations (drug protein molar ratio 0.001:1) and during short time incubations BP and PA selectively target different residues, Lys199 and Lys525, respectively. Molecular modeling showed that the selectivity was a function of noncovalent interaction before covalent modification. With increased exposure to higher concentrations of BP and PA, multiple epitopes were detected on albumin, demonstrating that the multiplicity of hapten formation is a function of time and concentration. More importantly, we have demonstrated direct evidence that PA is a hapten accounting for the diastereoisomeric BP antigen formation in albumin isolated from the blood of patients receiving penicillin. Furthermore, PA was found to be more potent than BP with respect to stimulation of T cells from patients with penicillin hypersensitivity, illustrating the functional relevance of diastereoisomeric hapten formation.

Cov-MS: A Community-Based Template Assay for Mass-Spectrometry-Based Protein Detection in SARS-CoV-2 Patients.

Rising population density and global mobility are among the reasons why pathogens such as SARS-CoV-2, the virus that causes COVID-19, spread so rapidly across the globe. The policy response to such pandemics will always have to include accurate monitoring of the spread, as this provides one of the few alternatives to total lockdown. However, COVID-19 diagnosis is currently performed almost exclusively by reverse transcription polymerase chain reaction (RT-PCR). Although this is efficient, automatable, and acceptably cheap, reliance on one type of technology comes with serious caveats, as illustrated by recurring reagent and test shortages. We therefore developed an alternative diagnostic test that detects proteolytically digested SARS-CoV-2 proteins using mass spectrometry (MS). We established the Cov-MS consortium, consisting of 15 academic laboratories and several industrial partners to increase applicability, accessibility, sensitivity, and robustness of this kind of SARS-CoV-2 detection. This, in turn, gave rise to the Cov-MS Digital Incubator that allows other laboratories to join the effort, navigate, and share their optimizations and translate the assay into their clinic. As this test relies on viral proteins instead of RNA, it provides an orthogonal and complementary approach to RT-PCR using other reagents that are relatively inexpensive and widely available, as well as orthogonally skilled personnel and different instruments. Data are available via ProteomeXchange with identifier PXD022550.

Safe nanoengineering and incorporation of transplant populations in a neurosurgical grade biomaterial, DuraGen PlusTM, for protected cell therapy applications.

High transplant cell loss is a major barrier to translation of stem cell therapy for pathologies of the brain and spinal cord. Encapsulated delivery of stem cells in biomaterials for cell therapy is gaining popularity but experimental research has overwhelmingly used laboratory grade materials unsuitable for human clinical use - representing a further barrier to clinical translation. A potential solution is to use neurosurgical grade materials routinely used in clinical protocols which have an established human safety profile. Here, we tested the ability of Duragen Plus™ - a clinical biomaterial used widely in neurosurgical duraplasty procedures, to support the growth and differentiation of neural stem cells- a major transplant population being tested in clinical trials for neurological pathology. Genetic engineering of stem cells yields augmented therapeutic cells, so we further tested the ability of the Duragen Plus™ matrix to support stem cells engineered using magnetofection technology and minicircle DNA vectors- a promising cell engineering approach we previously reported (Journal of Controlled Release, 2016 a &b). The safety of the nano-engineering approach was analysed for the first time using sophisticated data-independent analysis by mass spectrometry-based proteomics. We prove that the Duragen Plus™ matrix is a promising biomaterial for delivery of stem cell transplant populations, with no adverse effects on key regenerative parameters. This advanced cellular construct based on a combinatorial nano-engineering and biomaterial encapsulation approach, could therefore offer key advantages for clinical translation.

Proteomic investigation of urinary markers of carbon-tetrachloride-induced hepatic fibrosis in the Hanover Wistar rat.

Proteomic techniques such as two-dimensional gel electrophoresis (2-DGE) and mass spectrometry have become important tools for the identification of novel biomarkers of toxicity and disease. Ideally, such biomarkers need to be sensitive and organ specific, but, recently, it has become apparent that it would be an additional benefit to be able to measure biomarkers in samples obtained using non-invasive methods. The present study is concerned with the identification of novel urinary markers of hepatic fibrosis. In a carbon-tetrachloride-induced liver fibrosis rat model, analysis of urine by 2-DGE revealed an increase in the concentration of a number of proteins in animals with hepatic fibrosis. Using in-gel trypsin digest and nano-scale liquid chromatography combined with electrospray ionisation tandem mass spectrometry, protein spots were identified as copper/zinc superoxide dismutase, D: -dopachrome tautomerase, beta-2-microglobulin and neutrophil gelatinase associated lipocalin. These proteins are known to have important roles in the inflammatory response.

Analyzing Glycopeptide Isomers by Combining Differential Mobility Spectrometry with Electron- and Collision-Based Tandem Mass Spectrometry.

Differential mobility spectrometry (DMS) has been employed to separate isomeric species in several studies. Under the right conditions, factors such as separation voltage, temperature, the presence of chemical modifiers, and residence time can combine to provide unique signal channels for isomeric species. In this study, we examined a set of glycopeptide isomers, MUC5AC-3 and MUC5AC-13, which bear an N-acetyl-galactosamine (GalNAc) group on either threonine-3 or threonine-13. When analyzed as a mixture, the resulting MS and MS/MS spectra yield fragmentation patterns that cannot discern these convolved species. However, when DMS is implemented during the analysis of this mixture, two features emerge in the DMS ionogram representing the two glycopeptide isomers. In addition, by locking in DMS parameters at each feature, we could observe several low intensity CID fragments that contain the GalNAc functionality-specific amino acid residues - identifying the DMS separation of each isomer without standards. Besides conventional CID MS/MS, we also implemented electron-capture dissociation (ECD) after DMS separation, and clearly resolved both isomers with this fragmentation method, as well. The electron energy used in these ECD experiments could be tuned to obtain maximum sequence coverage for these glycopeptides; this was critical as these ions were present as doubly protonated species, which are much more difficult to fragment efficiently via electron-transfer dissociation (ETD). Overall, the combination of DMS with electron- or collision-based MS/MS methods provided enhanced separation and sequence coverage for these glycopeptide isomers. Graphical Abstract ᅟ.

Mass spectral enhanced detection of Ubls using SWATH acquisition: MEDUSA--simultaneous quantification of SUMO and ubiquitin-derived isopeptides.

Protein modification by ubiquitination and SUMOylation occur throughout the cell and are responsible for numerous cellular functions such as apoptosis, DNA replication and repair, and gene transcription. Current methods for the identification of such modifications using mass spectrometry predominantly rely upon tryptic isopeptide tag generation followed by database searching with in vitro genetic mutation of SUMO routinely required. We have recently described a novel approach to ubiquitin and SUMO modification detection based upon the diagnostic a' and b' ions released from the isopeptide tags upon collision-induced dissociation of reductively methylated Ubl isopeptides (RUbI) using formaldehyde. Here, we significantly extend those studies by combining data-independent acquisition (DIA) with alternative labeling reagents to improve diagnostic ion coverage and enable relative quantification of modified peptides from both MS and MS/MS signals. Model synthetic ubiquitin and SUMO-derived isopeptides were labeled with mTRAQ reagents (Δ0, Δ4, and Δ8) and subjected to LC-MS/MS with SWATH acquisition. Novel diagnostic ions were generated upon CID, which facilitated the selective detection of these modified peptides. Simultaneous MS-based and MS/MS-based relative quantification was demonstrated for both Ub and SUMO-derived isopeptides across three channels in a background of mTRAQ-labeled Escherichia coli digest.

Multiple reaction monitoring for quantitative biomarker analysis in proteomics and metabolomics.

The conventional pipeline for biomarker development involves a discovery phase, typically conducted by mass spectrometry (MS), followed by validation and clinical application, usually on an alternative platform, such as immunoassay. Whilst this approach is suitable for the development of single biomarkers, with the current drive towards larger panels of multiplexed biomarkers, the process becomes inefficient and costly. Consequently, the emphasis is now shifting towards performing full biomarker discovery, qualification and quantification on the same technology platform. The ease of multiplexing and ability to determine protein modifications makes MS an attractive alternative to antibody-based technologies. In addition, developments in quantitative MS, through the application of stable isotope labelling and scanning techniques, such as multiple reaction monitoring (MRM), have greatly enhanced both the specificity and sensitivity of MS-based assays to the point that they can rival immunoassay for some analytes. This review focuses on the application of MRM for quantitative MS analysis, particularly with respect to proteins and peptides.

A technically detailed and pragmatic protocol for quantitative serum proteomics using iTRAQ.

Blood is recognised as a highly important source of disease-related biomarkers, and proteomic approaches for identifying novel blood-borne biomarkers are in demand. The complexity and dynamic protein concentration range of plasma/serum however complicates the analysis process. A number of strategies for simplification of blood prior to proteomic analysis have been developed. In addition, methods for quantifying the levels of proteins in samples, such as isobaric tags for relative and absolute quantification (iTRAQ) are emerging. However, the successful application of these procedures is not always straightforward and technical hurdles must be overcome. Here we provide a technically detailed working protocol for iTRAQ-based quantification of serum proteins following immunodepletion of high abundance proteins. To improve the number of proteins identified and quantified we have introduced several modifications to the standard iTRAQ protocol. We report identifications of 217 proteins (5773 peptides) with a false discovery rate of 1% or 254 proteins with 95% confidence, respectively. Relative quantification data were obtained for 234 (95% confidence) serum proteins, including species present in the concentration range of tissue leakage factors. The samples described here relate to pancreatic cancer; however the protocol can be applied to serum from other control or disease types.

Glutathione-S-transferase pi as a model protein for the characterisation of chemically reactive metabolites.

Chemically reactive metabolites (CRMs) are thought to be responsible for a number of adverse drug reactions through modification of critical proteins. Methods that defined the chemistry of protein modification at an early stage would provide invaluable tools for drug safety assessment. Here, human GST pi (GSTP) was exploited as a model target protein to determine the chemical, biochemical and functional consequences of exposure to the hepatotoxic CRM of paracetamol (APAP), N-acetyl-p-benzoquinoneimine (NAPQI). Site-specific, dose-dependent modification of Cys47 in native and His-tagged GSTP was revealed by MS, and correlated with inhibition of glutathione (GSH) conjugating activity. In addition, the adaptation of iTRAQ labelling technology to define precisely the quantitative relationship between covalent modification and protein function is described. Multiple reaction monitoring (MRM)-MS of GSTP allowed high sensitivity detection of modified peptides at physiological levels of exposure. Finally, a bioengineered mutant cytochrome P450 with a broad spectrum of substrate specificities was used in an in vitro reaction system to bioactivate APAP: in this model, GSTP trapped the CRM and exhibited both reduced enzyme activity and site-specific modification of the protein. These studies provide the foundation for the development of novel test systems to predict the toxicological potential of CRMs produced by new therapeutic agents.

Comparative cytochrome P450 proteomics in the livers of immunodeficient mice using 18O stable isotope labeling.

Quantitative changes in cytochrome P450 (CYP) proteins involved in drug metabolism as a consequence of drug treatment are important parameters in predicting the fates and pharmacological consequences of xenobiotics and drugs. In this study we undertook comparative P450 proteomics using liver from control and 1,4-bis-2-(3,5-dichloropyridyloxybenzene) (TCPOBOP)-dosed mice. The method involved separation of microsomal proteins by SDS-PAGE, trypsin digestion, and postdigest 18O/16O labeling followed by nano-LC-MS/MS for peptide identification and LC-MS for relative quantification. Seventeen P450 proteins were identified from mouse liver of which 16 yielded data sufficient for relative quantification. All the P450s detected were unambiguously identified except the highly homologous CYP2A4/2A5. With the exception of CYP2A12, -2D10, and -2F2, the levels of all the P450s quantified were affected by treatment with TCPOBOP (3 mg/kg). CYP1A2, -2A4/5, -2B10, -2B20, -2C29, -2C37, -2C38, -3A11, and -39A1 were up-regulated, and CYP2C40, -2E1, -3A41, and -27A1 down-regulated. The response of CYP2B20 to stimulation has not been distinguished previously from that of CYP2B10 because of the poor discrimination between these two proteins (they share 87% sequence identity). Differential response to chemical stimulation by closely related members of the CYP2C subfamily was also observed.