Direct sub-atmospheric pressure ionization mass spectrometry: Evaporation/sublimation-driven ionization is amazing, fundamentally, and practically.

This paper covers direct sub-atmospheric pressure ionization mass spectrometry (MS). The discovery, applications, and mechanistic aspects of novel ionization processes for use in MS that are not based on the high-energy input from voltage, laser, and/or high temperature but on sublimation/evaporation within a region linking a higher to lower pressure and modulated by heat and collisions, are discussed, including how this new reality has guided a series of discoveries, instrument developments, and commercialization. A research focus, inter alia, is on how best to understand, improve, and use these novel ionization processes, which convert volatile and nonvolatile compounds from solids (sublimation) or liquids (evaporation) into gas-phase ions for analysis by MS providing reproducible, accurate, sensitive, and prompt results. Our perception on how these unprecedented versus traditional ionization processes/methods relate to each other, how they can be made to coexist on the same mass spectrometer, and an outlook on new and expanded applications (e.g., clinical, portable, fast, safe, and autonomous) is presented, and is based on ST's Opening lecture presentation at the Nordic Mass spectrometry Conference, Geilo, Norway, January 2023. Focus will be on matrix-assisted ionization (MAI) and solvent-assisted ionization (SAI) MS covering the period from 2010 to 2023; a potential paradigm shift in the making.

Human iN neuronal model of schizophrenia displays dysregulation of chromogranin B and related neuropeptide transmitter signatures.

Schizophrenia (SZ) is a serious mental illness and neuropsychiatric brain disorder with behavioral symptoms that include hallucinations, delusions, disorganized behavior, and cognitive impairment. Regulation of such behaviors requires utilization of neurotransmitters released to mediate cell-cell communication which are essential to brain functions in health and disease. We hypothesized that SZ may involve dysregulation of neurotransmitters secreted from neurons. To gain an understanding of human SZ, induced neurons (iNs) were derived from SZ patients and healthy control subjects to investigate peptide neurotransmitters, known as neuropeptides, which represent the major class of transmitters. The iNs were subjected to depolarization by high KCl in the culture medium and the secreted neuropeptides were identified and quantitated by nano-LC-MS/MS tandem mass spectrometry. Several neuropeptides were identified from schizophrenia patient-derived neurons, including chromogranin B (CHGB), neurotensin, and natriuretic peptide. Focusing on the main secreted CHGB neuropeptides, results revealed differences in SZ iNs compared to control iN neurons. Lower numbers of distinct CHGB peptides were found in the SZ secretion media compared to controls. Mapping of the peptides to the CHGB precursor revealed peptides unique to either SZ or control, and peptides common to both conditions. Also, the iNs secreted neuropeptides under both KCl and basal (no KCl) conditions. These findings are consistent with reports that chromogranin B levels are reduced in the cerebrospinal fluid and specific brain regions of SZ patients. These findings suggest that iNs derived from SZ patients can model the decreased CHGB neuropeptides observed in human SZ.

Identification of tyrosine sulfation in the variable region of a bispecific antibody and its effect on stability and biological activity.

Despite tyrosine sulfation being a relatively common post-translational modification (PTM) on the secreted proteins of higher eukaryotic organisms, there have been surprisingly few reports of this modification occurring in recombinant monoclonal antibodies (mAbs) expressed by mammalian cell lines and even less information regarding its potential impact on mAb efficacy and stability. This discrepancy is likely due to the extreme lability of this modification using many of the mass spectrometry methods typically used within the biopharmaceutical industry for PTM identification, as well as the possible misidentification as phosphorylation. Here, we identified sulfation on a single tyrosine residue located within the identical variable region sequence of a 2 + 1 bispecific mAbs heavy and heavy-heavy chains using a multi-enzymatic approach in combination with mass spectrometry analysis and examined its impact on binding, efficacy, and physical stability. Unlike previous reports, we found that tyrosine sulfation modestly decreased the mAb cell binding and T cell-mediated killing, primarily by increasing the rate of antigen disassociation as determined from surface plasmon resonance-binding experiments. We also found that, while this acidic modification had no significant impact on the mAb thermal stability, sulfation did modestly increase its rate of aggregation, presumably by lowering the mAb's colloidal stability as indicated by polyethylene glycol induced liquid-liquid phase separation experiments.

Selective Neutral pH Inhibitor of Cathepsin B Designed Based on Cleavage Preferences at Cytosolic and Lysosomal pH Conditions.

Cathepsin B is a cysteine protease that normally functions within acidic lysosomes for protein degradation, but in numerous human diseases, cathepsin B translocates to the cytosol having neutral pH where the enzyme activates inflammation and cell death. Cathepsin B is active at both the neutral pH 7.2 of the cytosol and the acidic pH 4.6 within lysosomes. We evaluated the hypothesis that cathepsin B may possess pH-dependent cleavage preferences that can be utilized for design of a selective neutral pH inhibitor by (1) analysis of differential cathepsin B cleavage profiles at neutral pH compared to acidic pH using multiplex substrate profiling by mass spectrometry (MSP-MS), (2) design of pH-selective peptide-7-amino-4-methylcoumarin (AMC) substrates, and (3) design and validation of Z-Arg-Lys-acyloxymethyl ketone (AOMK) as a selective neutral pH inhibitor. Cathepsin B displayed preferences for cleaving peptides with Arg in the P2 position at pH 7.2 and Glu in the P2 position at pH 4.6, represented by its primary dipeptidyl carboxypeptidase and modest endopeptidase activity. These properties led to design of the substrate Z-Arg-Lys-AMC having neutral pH selectivity, and its modification with the AOMK warhead to result in the inhibitor Z-Arg-Lys-AOMK. This irreversible inhibitor displays nanomolar potency with 100-fold selectivity for inhibition of cathepsin B at pH 7.2 compared to pH 4.6, shows specificity for cathepsin B over other cysteine cathepsins, and is cell permeable and inhibits intracellular cathepsin B. These findings demonstrate that cathepsin B possesses pH-dependent cleavage properties that can lead to development of a potent, neutral pH inhibitor of this enzyme.

Differential Neuropeptidomes of Dense Core Secretory Vesicles (DCSV) Produced at Intravesicular and Extracellular pH Conditions by Proteolytic Processing.

Neuropeptides mediate cell-cell signaling in the nervous and endocrine systems. The neuropeptidome is the spectrum of peptides generated from precursors by proteolysis within dense core secretory vesicles (DCSV). DCSV neuropeptides and contents are released to the extracellular environment where further processing for neuropeptide formation may occur. To assess the DCSV proteolytic capacity for production of neuropeptidomes at intravesicular pH 5.5 and extracellular pH 7.2, neuropeptidomics, proteomics, and protease assays were conducted using chromaffin granules (CG) purified from adrenal medulla. CG are an established model of DCSV. The CG neuropeptidome consisted of 1239 unique peptides derived from 15 proneuropeptides that were colocalized with 64 proteases. Distinct CG neuropeptidomes were generated at the internal DCSV pH of 5.5 compared to the extracellular pH of 7.2. Class-specific protease inhibitors differentially regulated neuropeptidome production involving aspartic, cysteine, serine, and metallo proteases. The substrate cleavage properties of CG proteases were assessed by multiplex substrate profiling by mass spectrometry (MSP-MS) that uses a synthetic peptide library containing diverse cleavage sites for endopeptidases and exopeptidases. Parallel inhibitor-sensitive cleavages for neuropeptidome production and peptide library proteolysis led to elucidation of six CG proteases involved in neuropeptidome production, represented by cathepsins A, B, C, D, and L and carboxypeptidase E (CPE). The MSP-MS profiles of these six enzymes represented the majority of CG proteolytic cleavages utilized for neuropeptidome production. These findings provide new insight into the DCSV proteolytic system for production of distinct neuropeptidomes at the internal CG pH of 5.5 and at the extracellular pH of 7.2.

Mutant Presenilin 1 Dysregulates Exosomal Proteome Cargo Produced by Human-Induced Pluripotent Stem Cell Neurons.

The accumulation and propagation of hyperphosphorylated tau (p-Tau) is a neuropathological hallmark occurring with neurodegeneration of Alzheimer's disease (AD). Extracellular vesicles, exosomes, have been shown to initiate tau propagation in the brain. Notably, exosomes from human-induced pluripotent stem cell (iPSC) neurons expressing the AD familial A246E mutant form of presenilin 1 (mPS1) are capable of inducing tau deposits in the mouse brain after in vivo injection. To gain insights into the exosome proteome cargo that participates in propagating tau pathology, this study conducted proteomic analysis of exosomes produced by human iPSC neurons expressing A246E mPS1. Significantly, mPS1 altered the profile of exosome cargo proteins to result in (1) proteins present only in mPS1 exosomes and not in controls, (2) the absence of proteins in the mPS1 exosomes which were present only in controls, and (3) shared proteins which were upregulated or downregulated in the mPS1 exosomes compared to controls. These results show that mPS1 dysregulates the proteome cargo of exosomes to result in the acquisition of proteins involved in the extracellular matrix and protease functions, deletion of proteins involved in RNA and protein translation systems along with proteasome and related functions, combined with the upregulation and downregulation of shared proteins, including the upregulation of amyloid precursor protein. Notably, mPS1 neuron-derived exosomes displayed altered profiles of protein phosphatases and kinases involved in regulating the status of p-tau. The dysregulation of exosome cargo proteins by mPS1 may be associated with the ability of mPS1 neuron-derived exosomes to propagate tau pathology.

An overview of biological applications and fundamentals of new inlet and vacuum ionization technologies.

RATIONALE: The developments of new ionization technologies based on processes previously unknown to mass spectrometry (MS) have gained significant momentum. Herein we address the importance of understanding these unique ionization processes, demonstrate the new capabilities currently unmet by other methods, and outline their considerable analytical potential. METHODS: The inlet and vacuum ionization methods of solvent-assisted ionization (SAI), matrix-assisted ionization (MAI), and laserspray ionization can be used with commercial and dedicated ion sources producing ions from atmospheric or vacuum conditions for analyses of a variety of materials including drugs, lipids, and proteins introduced from well plates, pipet tips and plate surfaces with and without a laser using solid or solvent matrices. Mass spectrometers from various vendors are employed. RESULTS: Results are presented highlighting strengths relative to ionization methods of electrospray ionization (ESI) and matrix-assisted laser desorption/ionization. We demonstrate the utility of multi-ionization platforms encompassing MAI, SAI, and ESI and enabling detection of what otherwise is missed, especially when directly analyzing mixtures. Unmatched robustness is achieved with dedicated vacuum MAI sources with mechanical introduction of the sample to the sub-atmospheric pressure (vacuum MAI). Simplicity and use of a wide array of matrices are attained using a conduit (inlet ionization), preferably heated, with sample introduction from atmospheric pressure. Tissue, whole blood, urine (including mouse, chicken, and human origin), bacteria strains and chemical on-probe reactions are analyzed directly and, especially in the case of vacuum ionization, without concern of carryover or instrument contamination. CONCLUSIONS: Examples are provided highlighting the exceptional analytical capabilities associated with the novel ionization processes in MS that reduce operational complexity while increasing speed and robustness, achieving mass spectra with low background for improved sensitivity, suggesting the potential of this simple ionization technology to drive MS into areas currently underserved, such as clinical and medical applications.

Resolving Isomers of Star-Branched Poly(Ethylene Glycols) by IMS-MS Using Multiply Charged Ions.

Ion mobility spectrometry (IMS) mass spectrometry (MS) centers on the ability to separate gaseous structures by size, charge, shape, and followed by mass-to-charge (m/z). For oligomeric structures, improved separation is hypothesized to be related to the ability to extend structures through repulsive forces between cations electrostatically bonded to the oligomers. Here we show the ability to separate differently branched multiply charged ions of star-branched poly(ethylene glycol) oligomers (up to 2000 Da) regardless of whether formed by electrospray ionization (ESI) charged solution droplets or from charged solid particles produced directly from a surface by matrix-assisted ionization. Detailed structural characterization of isomers of the star-branched compositions was first established using a home-built high-resolution ESI IMS-MS instrument. The doubly charged ions have well-resolved drift times, achieving separation of isomers and also allowing differentiation of star-branched versus linear oligomers. An IMS-MS "snapshot" approach allows visualization of architectural dispersity and (im)purity of samples in a straightforward manner. Analyses capabilities are shown for different cations and ionization methods using commercially available traveling wave IMS-MS instruments. Analyses directly from surfaces using the new ionization processes are, because of the multiply charging, not only associated with the benefits of improved gas-phase separations, relative to that of ions produced by matrix-assisted laser desorption/ionization, but also provide the potential for spatially resolved measurements relative to ESI and other ionization methods.

Comparison of gaseous ubiquitin ion structures obtained from a solid and solution matrix using ion mobility spectrometry/mass spectrometry.

RATIONALE: Examining surface protein conformations, and especially achieving this with spatial resolution, is an important goal. The recently discovered ionization processes offer spatial-resolution measurements similar to matrix-assisted laser desorption/ionization (MALDI) and produce charge states similar to electrospray ionization (ESI) extending higher-mass protein applications directly from surfaces on high-performance mass spectrometers. Studying a well-interrogated protein by ion mobility spectrometry-mass spectrometry (IMS-MS) to access effects on structures using a solid vs. solvent matrix may provide insights. METHODS: Ubiquitin was studied by IMS-MS using new ionization processes with commercial and homebuilt ion sources and instruments (Waters SYNAPT G2(S)) and homebuilt 2 m drift-tube instrument; MS™ sources). Mass-to-charge and drift-time (td )-measurements are compared for ubiquitin ions obtained by inlet and vacuum ionization using laserspray ionization (LSI), matrix- (MAI) and solvent-assisted ionization (SAI), respectively, and compared with those from ESI under conditions that are most comparable. RESULTS: Using the same solution conditions with SYNAPT G2(S) instruments, td -distributions of various ubiquitin charge states from MAI, LSI, and SAI are similar to those from ESI using a variety of solvents, matrices, extraction voltages, a laser, and temperature only, showing subtle differences in more compact features within the elongated distribution of structures. However, on a homebuilt drift-tube instrument, within the elongated distribution of structures, both similar and different td -distributions are observed for ubiquitin ions obtained by MAI and ESI. MAI-generated ions are frequently narrower in their td -distributions. CONCLUSIONS: Direct comparisons between ESI and the new ionization methods operational directly from surfaces suggest that the protein in its solution structure prior to exposure to the ionization event is either captured (frozen out) at the time of crystallization, or that the protein in the solid matrix is associated with sufficient solvent to maintain the solution structure, or, alternatively, that the observed structures are those related to what occurs in the gas phase with ESI- or MAI-generated ions and not with the solution structures.

Proteomic Modulation in the Dorsal Spinal Cord Following Spinal Cord Stimulation Therapy in an In Vivo Neuropathic Pain Model.

OBJECTIVES: Spinal cord stimulation (SCS) provides relief for patients suffering from chronic neuropathic pain although its mechanism may not be as dependent on electrical interference as classically considered. Recent evidence has been growing regarding molecular changes that are induced by SCS as being a key player in reversing the pain process. Here, we observed the effect of SCS on altering protein expression in spinal cord tissue using a proteomic analysis approach. METHODS: A microlead was epidurally implanted following induction of an animal neuropathic pain model. After the model was established, stimulation was applied for 72 hours continuously followed by tissue collection and proteomic analysis via tandem mass spectroscopy. Identified proteins were run through online data bases for protein identification and classification of biological processes. RESULTS: A significant improvement in mechanical sensitivity was observed following 48 hours of SCS therapy. Proteomic analysis identified 5840 proteins, of which 155 were significantly affected by SCS. Gene ontology data bases indicated that a significant number of proteins were associated to stress response, oxidation/reduction, or extracellular matrix pathways. Additionally, many of the proteins identified also play a role in neuron-glial interactions and are involved in nociception. CONCLUSIONS: The development of an injury unbalances the proteome of the local neural tissue, neurons, and glial cells, and shifts the proteomic profile to a pain producing state. This study demonstrates the reversal of the injury-induced proteomic state by applying conventional SCS therapy. Additional studies looking at variations in electrical parameters are needed to optimize SCS.

High-Resolution Mass Spectrometry-Based Approaches for the Detection and Quantification of Peptidase Activity in Plasma.

Proteomic technologies have identified 234 peptidases in plasma but little quantitative information about the proteolytic activity has been uncovered. In this study, the substrate profile of plasma proteases was evaluated using two nano-LC-ESI-MS/MS methods. Multiplex substrate profiling by mass spectrometry (MSP-MS) quantifies plasma protease activity in vitro using a global and unbiased library of synthetic peptide reporter substrates, and shotgun peptidomics quantifies protein degradation products that have been generated in vivo by proteases. The two approaches gave complementary results since they both highlight key peptidase activities in plasma including amino- and carboxypeptidases with different substrate specificity profiles. These assays provide a significant advantage over traditional approaches, such as fluorogenic peptide reporter substrates, because they can detect active plasma proteases in a global and unbiased manner, in comparison to detecting select proteases using specific reporter substrates. We discovered that plasma proteins are cleaved by endoproteases and these peptide products are subsequently degraded by amino- and carboxypeptidases. The exopeptidases are more active and stable in plasma and therefore were found to be the most active proteases in the in vitro assay. The protocols presented here set the groundwork for studies to evaluate changes in plasma proteolytic activity in shock.

Highly multiplexed quantitative proteomic and phosphoproteomic analyses in vascular smooth muscle cell dedifferentiation.

Restenosis, re-narrowing of arterial lumen following intervention for cardiovascular disease, remains a major issue limiting the long-term therapeutic efficacy of treatment. The signaling molecules, TGFß (transforming growth factor-beta) and Smad3, play important roles in vascular restenosis, but very little is yet known about the down-stream dynamics in global protein expression and phosphorylation. Here, we develop a highly multiplexed quantitative proteomic and phosphoproteomic strategy employing 12-plex N,N-dimethyl leucine (DiLeu) isobaric tags and The DiLeu Tool software to globally assess protein expression and phosphorylation changes in smooth muscle cells (SMCs) treated with TGFß/Smad3 and/or SDF-1α (stromal cell-derived factor). A total of 4086 proteins were quantified in the combined dataset of proteome and phosphoproteome across 12-plex DiLeu-labeled SMC samples. 2317 localized phosphorylation sites were quantified, corresponding to 1193 phosphoproteins. TGFß/Smad3 induced up-regulation of 40 phosphosites and down-regulation of 50 phosphosites, and TGFß/Smad3-specific SDF-1α exclusively facilitated up-regulation of 27 phosphosites and down-regulation of 47 phosphosites. TGFß/Smad3 inhibited the expression of contractile-associated proteins including smooth muscle myosin heavy chain, calponin, cardiac muscle alpha-actin, and smooth muscle protein 22α. Gene ontology and pathway enrichment analysis revealed that elevated TGFß/Smad3 activated cell proliferation and TGFß signaling pathway, sequentially stimulating phosphorylation of CXCR4 (C-X-C chemokine receptor 4). SDF-1α/CXCR4 activated extracellular signal-regulating kinase signaling pathway and facilitated the expression of synthetic marker, osteopontin, which was validated through targeted analysis. These findings provide new insights into the mechanisms of TGFß regulated SMC dedifferentiation, as well as new avenues for designing effective therapeutics for vascular disease.

Dysregulation of Exosome Cargo by Mutant Tau Expressed in Human-induced Pluripotent Stem Cell (iPSC) Neurons Revealed by Proteomics Analyses.

Accumulation and propagation of hyperphosphorylated Tau (p-Tau) is a common neuropathological hallmark associated with neurodegeneration of Alzheimer's disease (AD), frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), and related tauopathies. Extracellular vesicles, specifically exosomes, have recently been demonstrated to participate in mediating Tau propagation in brain. Exosomes produced by human induced pluripotent stem cell (iPSC)-derived neurons expressing mutant Tau (mTau), containing the P301L and V337M Tau mutations of FTDP-17, possess the ability to propagate p-Tau pathology after injection into mouse brain. To gain an understanding of the mTau exosome cargo involved in Tau pathogenesis, these pathogenic exosomes were analyzed by proteomics and bioinformatics. The data showed that mTau expression dysregulates the exosome proteome to result in 1) proteins uniquely present only in mTau, and not control exosomes, 2) the absence of proteins in mTau exosomes, uniquely present in control exosomes, and 3) shared proteins which were significantly upregulated or downregulated in mTau compared with control exosomes. Notably, mTau exosomes (not control exosomes) contain ANP32A (also known as I1PP2A), an endogenous inhibitor of the PP2A phosphatase which regulates the phosphorylation state of p-Tau. Several of the mTau exosome-specific proteins have been shown to participate in AD mechanisms involving lysosomes, inflammation, secretases, and related processes. Furthermore, the mTau exosomes lacked a substantial portion of proteins present in control exosomes involved in pathways of localization, vesicle transport, and protein binding functions. The shared proteins present in both mTau and control exosomes represented exosome functions of vesicle-mediated transport, exocytosis, and secretion processes. These data illustrate mTau as a dynamic regulator of the biogenesis of exosomes to result in acquisition, deletion, and up- or downregulation of protein cargo to result in pathogenic mTau exosomes capable of in vivo propagation of p-Tau neuropathology in mouse brain.

The Proteasome as a Drug Target in the Metazoan Pathogen, Schistosoma mansoni.

Proteases are fundamental to successful parasitism, including that of the schistosome flatworm parasite, which causes the disease schistosomiasis in 200 million people worldwide. The proteasome is receiving attention as a potential drug target for treatment of a variety of infectious parasitic diseases, but it has been understudied in the schistosome. Adult Schistosoma mansoni were incubated with 1 µM concentrations of the proteasome inhibitors bortezomib, carfilzomib, and MG132. After 24 h, bortezomib and carfilzomib decreased worm motility by more than 85% and endogenous proteasome activity by >75%, and after 72 h, they increased caspase activity by >4.5-fold. The association between the engagement of the proteasome target and the phenotypic and biochemical effects recorded encouraged the chromatographic enrichment of the S. mansoni proteasome (Sm20S). Activity assays with fluorogenic proteasome substrates revealed that Sm20S contains caspase-type (ß1), trypsin-type (ß2), and chymotrypsin-type (ß5) activities. Sm20S was screened with 11 peptide epoxyketone inhibitors derived from the marine natural product carmaphycin B. Analogue 17 was 27.4-fold less cytotoxic to HepG2 cells than carmaphycin B and showed equal potency for the ß5 subunits of Sm20S, human constitutive proteasome, and human immunoproteasome. However, this analogue was 13.2-fold more potent at targeting Sm20S ß2 than it was at targeting the equivalent subunits of the human enzymes. Furthermore, 1 µM 17 decreased both worm motility and endogenous Sm20S activity by more than 90% after 24 h. We provide direct evidence of the proteasome's importance to schistosome viability and identify a lead for which future studies will aim to improve the potency, selectivity, and safety.

In Depth Quantification of Extracellular Matrix Proteins from Human Pancreas.

Extracellular matrix (ECM) is an important component of the pancreatic microenvironment which regulates ß cell proliferation, differentiation, and insulin secretion. Protocols have recently been developed for the decellularization of the human pancreas to generate functional scaffolds and hydrogels. In this work, we characterized human pancreatic ECM composition before and after decellularization using isobaric dimethylated leucine (DiLeu) labeling for relative quantification of ECM proteins. A novel correction factor was employed in the study to eliminate the bias introduced during sample preparation. In comparison to the commonly employed sample preparation methods (urea and FASP) for proteomic analysis, a recently developed surfactant and chaotropic agent assisted sequential extraction/on pellet digestion (SCAD) protocol has provided an improved strategy for ECM protein extraction of human pancreatic ECM matrix. The quantitative proteomic results revealed the preservation of matrisome proteins while most of the cellular proteins were removed. This method was compared with a well-established label-free quantification (LFQ) approach which rendered similar expressions of different categories of proteins (collagens, ECM glycoproteins, proteoglycans, etc.). The distinct expression of ECM proteins was quantified comparing adult and fetal pancreas ECM, shedding light on the correlation between matrix composition and postnatal ß cell maturation. Despite the distinct profiles of different subcategories in the native pancreas, the distribution of matrisome proteins exhibited similar trends after the decellularization process. Our method generated a large data set of matrisome proteins from a single tissue type. These results provide valuable insight into the possibilities of constructing a bioengineered pancreas. It may also facilitate better understanding of the potential roles that matrisome proteins play in postnatal ß cell maturation.

A Commensal Dipeptidyl Aminopeptidase with Specificity for N-Terminal Glycine Degrades Human-Produced Antimicrobial Peptides in Vitro.

Proteases within the C1B hydrolase family are encoded by many organisms. We subjected a putative C1B-like cysteine protease secreted by the human gut commensal Parabacteroides distasonis to mass spectrometry-based substrate profiling to find preferred peptide substrates. The P. distasonis protease, which we termed Pd_dinase, has a sequential diaminopeptidase activity with strong specificity for N-terminal glycine residues. Using the substrate sequence information, we verified the importance of the P2 glycine residue with a panel of fluorogenic substrates and calculated kcat and KM for the dipeptide glycine-arginine-AMC. A potent and irreversible dipeptide inhibitor with a C-terminal acyloxymethyl ketone warhead, glycine-arginine- AOMK, was then synthesized and demonstrated that the Pd_dinase active site requires a free N-terminal amine for potent and rapid inhibition. We next determined the homohexameric Pd_dinase structure in complex with glycine-arginine- AOMK and uncovered unexpected active site features that govern the strict substrate preferences and differentiate this protease from members of the C1B and broader papain-like C1 protease families. We finally showed that Pd_dinase hydrolyzes several human antimicrobial peptides and therefore posit that this P. distasonis enzyme may be secreted into the extracellular milieu to assist in gut colonization by inactivation of host antimicrobial peptides.

Diversity of Neuropeptide Cell-Cell Signaling Molecules Generated by Proteolytic Processing Revealed by Neuropeptidomics Mass Spectrometry.

Neuropeptides are short peptides in the range of 3-40 residues that are secreted for cell-cell communication in neuroendocrine systems. In the nervous system, neuropeptides comprise the largest group of neurotransmitters. In the endocrine system, neuropeptides function as peptide hormones to coordinate intercellular signaling among target physiological systems. The diversity of neuropeptide functions is defined by their distinct primary sequences, peptide lengths, proteolytic processing of pro-neuropeptide precursors, and covalent modifications. Global, untargeted neuropeptidomics mass spectrometry is advantageous for defining the structural features of the thousands to tens of thousands of neuropeptides present in biological systems. Defining neuropeptide structures is the basis for defining the proteolytic processing pathways that convert pro-neuropeptides into active peptides. Neuropeptidomics has revealed that processing of pro-neuropeptides occurs at paired basic residues sites, and at non-basic residue sites. Processing results in neuropeptides with known functions and generates novel peptides representing intervening peptide domains flanked by dibasic residue processing sites, identified by neuropeptidomics. While very short peptide products of 2-4 residues are predicted from pro-neuropeptide dibasic processing sites, such peptides have not been readily identified; therefore, it will be logical to utilize metabolomics to identify very short peptides with neuropeptidomics in future studies. Proteolytic processing is accompanied by covalent post-translational modifications (PTMs) of neuropeptides comprising C-terminal amidation, N-terminal pyroglutamate, disulfide bonds, phosphorylation, sulfation, acetylation, glycosylation, and others. Neuropeptidomics can define PTM features of neuropeptides. In summary, neuropeptidomics for untargeted, global analyses of neuropeptides is essential for elucidation of proteases that generate diverse neuropeptides for cell-cell signaling. Graphical Abstract ᅟ.

Phosphopeptidomics Reveals Differential Phosphorylation States and Novel SxE Phosphosite Motifs of Neuropeptides in Dense Core Secretory Vesicles.

Neuropeptides are vital for cell-cell communication and function in the regulation of the nervous and endocrine systems. They are generated by post-translational modification (PTM) steps resulting in small active peptides generated from prohormone precursors. Phosphorylation is a significant PTM for the bioactivity of neuropeptides. From the known diversity of distinct neuropeptide functions, it is hypothesized that the extent of phosphorylation varies among different neuropeptides. To assess this hypothesis, neuropeptide-containing dense core secretory vesicles from bovine adrenal medullary chromaffin cells were subjected to global phosphopeptidomics analyses by liquid chromatography (LC)-mass spectrometry (MS/MS). Phosphopeptides were identified directly by LC-MS/MS and indirectly by phosphatase treatment followed by LC-MS/MS. The data identified numerous phosphorylated peptides derived from neuropeptide precursors such as chromogranins, secretogranins, proenkephalin and pro-NPY. Phosphosite occupancies were observed at high and low levels among identified peptides and many of the high occupancy phosphopeptides represent prohormone-derived peptides with currently unknown bioactivities. Peptide sequence analyses demonstrated SxE as the most prevalent phosphorylation site motif, corresponding to phosphorylation sites of the Fam20C protein kinase known to be present in the secretory pathway. The range of high to low phosphosite occupancies for neuropeptides demonstrates cellular regulation of neuropeptide phosphorylation. Graphical Abstract ᅟ.

Coupling matrix-assisted ionization with high resolution mass spectrometry and electron transfer dissociation to characterize intact proteins and post-translational modifications.

Matrix-assisted ionization (MAI) is a recently developed ionization technique that produces multiply charged ions on either electrospray ionization (ESI) or matrix-assisted laser desorption/ionization (MALDI) platform without the need of high voltage or laser ablation. In this study, MAI has been coupled to a high resolution accurate mass (HRAM) hybrid instrument, the Orbitrap Elite mass spectrometer, with electron transfer dissociation (ETD) module for fast peptide and intact protein characterization. The softness of MAI process preserves labile post-translational modifications (PTM) and allows fragmentation and localization by ETD. Moreover, MAI on ESI platform allows rapid sample preparation and analysis (~ 1 min/sample) due to the easiness of sample introduction. It significantly improves the throughput compared to ESI direct infusion and MAI on MALDI platform, which usually takes more than 10 min/sample. Intact protein standards, protein mixtures, and neural tissue extracts have been characterized using this instrument platform with both full MS and MS/MS (CID, HCD, and ETD) analyses. Furthermore, the performances of ESI, MALDI, and MAI on both platforms have been tested to provide a systematic comparison among these techniques. With improved ETD performance and PTM analysis capabilities, we anticipate that the HRAM MAI-MS with ETD module will offer greater utilities in large molecule characterization with enhanced speed and coverage. These advancements will enable promising applications in bottom-up and top-down protein analyses. Graphical abstract Matrix-assisted ionization (MAI) for characterizing intact proteins and post-translational modifications with representative mass spectra from intact proteins.

Mass Defect-Based N,N-Dimethyl Leucine Labels for Quantitative Proteomics and Amine Metabolomics of Pancreatic Cancer Cells.

Mass spectrometry-based stable isotope labeling has become a key technology for protein and small-molecule analyses. We developed a multiplexed quantification method for simultaneous proteomics and amine metabolomics analyses via nano reversed-phase liquid chromatography-tandem mass spectrometry (nanoRPLC-MS/MS), called mass defect-based N,N-dimethyl leucine (mdDiLeu) labeling. The duplex mdDiLeu reagents were custom-synthesized with a mass difference of 20.5 mDa, arising from the subtle variation in nuclear binding energy between the two DiLeu isotopologues. Optimal MS resolving powers were determined to be 240K for labeled peptides and 120K for labeled metabolites on the Orbitrap Fusion Lumos instrument. The mdDiLeu labeling does not suffer from precursor interference and dynamic range compression, providing excellent accuracy for MS1-centric quantification. Quantitative information is only revealed at high MS resolution without increasing spectrum complexity and overlapping isotope distribution. Chromatographic performance of polar metabolites was dramatically improved by mdDiLeu labeling with modified hydrophobicity, enhanced ionization efficiency, and picomole levels of detection limits. Paralleled proteomics and amine metabolomics analyses using mdDiLeu were systematically evaluated and then applied to pancreatic cancer cells.