Project description:Advances in software and high resolution, high mass accuracy mass spectrometers have expanded their functionality beyond traditional data dependent acquisition (DDA) methods. Using a single platform, an orthogonal quadrupole time-of flight (QqTOF) mass spectrometer, the TripleTOF 5600, we have investigated the feasibility of implementing large-scale targeted quantitative assays, derived from discovery type data sets, using scheduled, high resolution multiple reaction monitoring (sMRM-HR) mass spectrometry. We assessed the selectivity and reproducibility of MRM-HR, also referred to as parallel reaction monitoring (PRM), measuring standard peptide concentration curves as well as system suitability assays. We specifically compared the robustness and accuracy of MRM-HR assays to traditional SRM workflows on triple quadrupole instruments. To determine the utility of sMRM-HR for large-scale targeted quantitative assays, we retention time scheduled over 500 peptides in a single LC-MS acquisition. High resolution and high mass accuracy of the full scan MS/MS spectra resulted in sufficient selectivity to monitor numerous MS/MS fragment ions per analyte precursor and provided flexibility for post-acquisition assay refinement and optimization. To demonstrate its applicability to biological samples, whole cell lysates from several E. coli wild-type and mutant strains were quantitatively assayed by sMRM-HR to confirm a previously generated candidate list of differentially expressed proteins. The ease of developing and implementing sMRM-HR assays derived directly from DDA discovery workflows on the same high resolution instrument platform facilitates downstream validation studies targeting many peptides for MS/MS level quantitation. This work provides a robust MRM-HR workflow for rapidly moving from discovery analysis to large-scale, multiplexed, targeted quantitation.
Project description:Lactoferrin is an 80 kDa bilobal, iron binding glycoprotein which is primarily antimicrobial in nature. The hydrolysis of lactoferrin by various proteases in the gut produces several functional fragments of lactoferrin which have varying molecular sizes and properties. Here, bovine lactoferrin has been hydrolyzed by trypsin, the major enzyme present in the gut, to produce three functional molecules of sizes approximately 21 kDa, 38 kDa and 45 kDa. The molecules have been purified using ion exchange and gel filtration chromatography and identified using N-terminal sequencing, which reveals that while the 21 kDa molecule corresponds to the N2 domain (21LF), the 38 kDa represents the whole C-lobe (38LF) and the 45 kDa is a portion of N1 domain of N-lobe attached to the C-lobe (45LF). The iron binding and release properties of 21LF, 38LF and 45LF have been studied and compared. The sequence and structure analysis of the portions of the excision sites of LF from various species have been done. The antibacterial properties of these three molecules against bacterial strains, Streptococcus pyogenes, Escherichia coli, Yersinia enterocolitica and Listeria monocytogenes were investigated. The antifungal action of the molecules was also evaluated against Candida albicans. This is the first report on the antimicrobial actions of the trypsin cleaved functional molecules of lactoferrin from any species.
Project description:Acetaminophen (N-acetyl-p-aminophenol; APAP) is a mild analgesic and antipyretic used commonly worldwide. Although APAP is considered a safe and effective over-the-counter medication, it is also the leading cause of drug-induced acute liver failure. Its hepatotoxicity has been linked to the covalent binding of its reactive metabolite, N-acetyl p-benzoquinone imine (NAPQI), to proteins. The aim of this in vivo study was to identify APAP-protein targets in both rat and mouse liver, and to compare the results from both species, using bottom-up proteomics and targeted multiple reaction monitoring (MRM) experiments. Livers from rats and mice, treated with APAP, were homogenized and digested by trypsin. Digests were then fractionated by mixed-mode solid-phase extraction prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS) using scheduled multiple reaction monitoring (MRM) acquisition. The targeted assays were optimized based on high-resolution MS/MS data from information-dependent acquisition (IDA) using control liver homogenates treated with a custom alkylating reagent forming a positional isomer of the APAP modification on all cysteine residues, in order to build an in-house modified peptide database for targeted analysis. A list of putative in vivo targets of APAP were screened from previous in vitro studies, data-dependent high-resolution MS/MS analyses of liver digests, as well as selected proteins from the target protein database (TPDB), an online resource which references previous reports of proteins found to be modified by acetaminophen. Multiple protein targets of APAP in each species were found, while confirming modification sites.
Project description:The lack of consensus sequence, common core structure, and universal endoglycosidase for the release of O-linked oligosaccharides makes O-glycosylation more difficult to tackle than N-glycosylation. Structural elucidation by mass spectrometry is usually inconclusive as the CID spectra of most glycopeptides are dominated by carbohydrate-related fragments, preventing peptide identification. In addition, O-linked structures also undergo a gas-phase rearrangement reaction, which eliminates the sugar without leaving a telltale sign at its former attachment site. In the present study we report the enrichment and mass spectrometric analysis of proteins from bovine serum bearing Galbeta1-3GalNAcalpha (mucin core-1 type) structures and the analysis of O-linked glycopeptides utilizing electron transfer dissociation and high resolution, high mass accuracy precursor ion measurements. Electron transfer dissociation (ETD) analysis of intact glycopeptides provided sufficient information for the identification of several glycosylation sites. However, glycopeptides frequently feature precursor ions of low charge density (m/z > approximately 850) that will not undergo efficient ETD fragmentation. Exoglycosidase digestion was utilized to reduce the mass of the molecules while retaining their charge. ETD analysis of species modified by a single GalNAc at each site was significantly more successful in the characterization of multiply modified molecules. We report the unambiguous identification of 21 novel glycosylation sites. We also detail the limitations of the enrichment method as well as the ETD analysis.
Project description:We investigated the combined sensitivity of micro-flow liquid chromatography with a ZenoTOF mass spectrometer for high throughput proteomic and phosphoproteomic analysis of rat tissues. Comparing the proteomes acquired using data-independent acquisition (DIA) on the ZenoTOF 7600 with the previous generation TripleTOF 6600, more proteins were quantified using a fifth of the sample load and a third of the instrument time. Zeno SWATH data was evaluated using replicate injections of rat organ digests to compare FragPipe and DIA-NN computational pipelines. FragPipe identified more proteins in 7 of the 8 rat organs, with an extra 12% and 17% observed in heart and muscle tissue respectively. The number of identified peptides per protein were higher with FragPipe and the precision of missing values across replicate injections was more consistent. Single-shot phosphopeptide enrichment from 100 µg rat tissue, without fractionation, was acquired using data-dependent acquisition (DDA) on both instruments. A total of 5,108 phosphosites were quantified with a negligible increase in phosphosites found using the ZenoTOF 7600 relative to the 6600. Using DIA on the ZenoTOF, 8,013 phosphosites were quantified using Spectronaut.
Project description:Establishment and maintenance of pregnancy is dependent on progesterone synthesized by the corpus luteum (CL). The CL is known for the prominent presence of intracellular lipid droplets (LDs). However relatively little is known about the composition and function of these luteal LDs. Our objective was to identify the lipid composition of LDs from fully functional bovine CLs. Luteal LDs were isolated by flotation through a discontinuous sucrose gradient, lipids were then extracted using a standard Bligh and Dyer protocol, dried, and sent to Avanti Polar Lipids for lipidomics analysis. The samples were provided for lipidomic profiling of free sterols, cholesteryl esters, triglycerides, diacylglycerols, phospholipids, and sphingolipids. Molecular species were resolved by reversed-phase liquid chromatography in the presence of class and sub-class specific internal standard compounds added to each sample. The compounds were detected by tandem mass spectrometry (MS/MS) with scheduled multiple reaction monitoring (MRM) for mass-specific fragment ions according to the lipid class and molecular weight of the compound. Quantification of cholesterol, cholesteryl esters, triglycerides, and diglycerides were directly calculated with standards and internal standards from calibration response curves. The remaining lipid species were semi-quantization using the integrated area of each analyte’s MRM peak, divided by the appropriate internal standard peak area, and multiplied by the standard’s known concentration. Lipid concentrations were normalized to the corresponding protein concentration of each sample and as a mol % relative to total lipids or within each lipid class. Isolated luteal LDs were composed primarily of triglyceride (88%, mol% of lipid class to total lipids). Other neutral lipids included diacylglycerol, 2.9%; and cholesteryl esters, 1.5%. Polar lipids were primarily composed of phosphatidylcholine (3.1%), sphingomyelin (1.5%), phosphatidylinositol (0.9%), phosphatidylethanolamine (0.8%) and phosphatidylserine (0.4%). A number of other minor lipids representing less than 0.32% of the total lipid pool were also detected including phosphatidylglycerol, lysophospholipids, ceramides, and glycosylated ceramides. Lipid composition of bovine luteal LDs are distinct from LDs isolated from other tissues and in other species.
Project description:1. The electrophoretically fast (F) and slow (S) fragments obtained by tryptic cleavage of bovine iron-saturated transferrin differed in carbohydrate content and peptide 'maps'. 2. A fragment capable of binding one Fe3+ ion per molecule was isolated after brief tryptic digestion of bovine apotransferrin and shown closely to resemble the S fragment obtained from the iron-saturated protein. 3. Fragments F and S are probably derived from the N- and C-terminal halves of the transferrin molecule respectively. 4. Bovine transferrin could donate iron to rabbit reticulocytes, but the monoferric fragments possessed little iron-donating ability.
Project description:Ongoing immunomodulatory strategies in tumors characterized by an overall hot immune phenotype may improve prognosis of patients with non-small cell lung cancer (NSCLC). Our objective was to develop a reliable and stable scoring system for the identification of immunologically hot NSCLC and to evaluate its association with response to immunotherapies. A Hot Oral Tumor (HOT) score was developed using data from The Cancer Genome Atlas. HOT score was computed in 82 patients with NSCLC treated with second-line immunotherapy targeting PD-1/PD-L1. High HOT score was associated with a statistically significant improved clinical outcome.
Project description:Ongoing immunomodulatory strategies in tumors characterized by an overall hot immune phenotype may improve prognosis of head and neck squamous cell carcinomas (HNSCC). Our objective was to develop a reliable and stable scoring system for the identification of immunologically hot HNSCC and to evaluate its association with response to immunotherapies. A Hot Oral Tumor (HOT) score was developed using data from The Cancer Genome Atlas. HOT score was computed in 102 patients with HNSCC treated with immunotherapy targeting PD-1/PD-L1 in the context of clinical trials. High HOT score was associated with a statistically significant improved clinical outcome.