Quantitative proteomics reveals insights into the assembly of IFT trains and ciliary assembly.

Intraflagellar transport (IFT) is required for ciliary assembly. The IFT machinery comprises the IFT motors kinesin-2 and IFT dynein plus IFT-A and IFT-B complexes, which assemble into IFT trains in cilia. To gain mechanistic understanding of IFT and ciliary assembly, here, we performed an absolute quantification of IFT machinery in Chlamydomonas reinhardtii cilium. There are ∼756, ∼532, ∼276 and ∼350 molecules of IFT-B, IFT-A, IFT dynein and kinesin-2, respectively, per cilium. The amount of IFT-B is sufficient to sustain rapid ciliary growth in terms of tubulin delivery. The stoichiometric ratio of IFT-B:IFT-A:dynein is ∼3:2:1 whereas the IFT-B:IFT-A ratio in an IFT dynein mutant is 2:1, suggesting that there is a plastic interaction between IFT-A and IFT-B that can be influenced by IFT dynein. Considering diffusion of kinesin-2 during retrograde IFT, it is estimated that one kinesin-2 molecule drives eight molecules of IFT-B during anterograde IFT. These data provide new insights into the assembly of IFT trains and ciliary assembly.

Proteomic quantification of receptor tyrosine kinases involved in the development and progression of colorectal cancer liver metastasis.

Introduction: Alterations in expression and activity of human receptor tyrosine kinases (RTKs) are associated with cancer progression and in response to therapeutic intervention. Methods: Thus, protein abundance of 21 RTKs was assessed in 15 healthy and 18 cancerous liver samples [2 primary and 16 colorectal cancer liver metastasis (CRLM)] matched with non-tumorous (histologically normal) tissue, by a validated QconCAT-based targeted proteomic approach. Results: It was demonstrated, for the first time, that the abundance of EGFR, INSR, VGFR3 and AXL, is lower in tumours relative to livers from healthy individuals whilst the opposite is true for IGF1R. EPHA2 was upregulated in tumour compared with histologically normal tissue surrounding it. PGFRB levels were higher in tumours relative to both histologically normal tissue surrounding tumour and tissues taken from healthy individuals. The abundances of VGFR1/2, PGFRA, KIT, CSF1R, FLT3, FGFR1/3, ERBB2, NTRK2, TIE2, RET, and MET were, however, comparable in all samples. Statistically significant, but moderate correlations were observed (Rs > 0.50, p < 0.05) for EGFR with INSR and KIT. FGFR2 correlated with PGFRA and VGFR1 with NTRK2 in healthy livers. In non-tumorous (histologically normal) tissues from cancer patients, there were correlations between TIE2 and FGFR1, EPHA2 and VGFR3, FGFR3 and PGFRA (p < 0.05). EGFR correlated with INSR, ERBB2, KIT and EGFR, and KIT with AXL and FGFR2. In tumours, CSF1R correlated with AXL, EPHA2 with PGFRA, and NTRK2 with PGFRB and AXL. Sex, liver lobe and body mass index of donors had no impact on the abundance of RTKs, although donor age showed some correlations. RET was the most abundant of these kinases in non-tumorous tissues (~35%), while PGFRB was the most abundant RTK in tumours (~47%). Several correlations were also observed between the abundance of RTKs and proteins relevant to drug pharmacokinetics (enzymes and transporters). Discussion: DiscussionThis study quantified perturbation to the abundance of several RTKs in cancer and the value generated in this study can be used as input to systems biology models defining liver cancer metastases and biomarkers of its progression.

FastCAT Accelerates Absolute Quantification of Proteins Using Multiple Short Nonpurified Chimeric Standards.

Absolute (molar) quantification of clinically relevant proteins determines their reference values in liquid and solid biopsies. The FastCAT (for Fast-track QconCAT) method employs multiple short (<50 kDa), stable-isotope labeled chimeric proteins (CPs) composed of concatenated quantotypic (Q)-peptides representing the quantified proteins. Each CP also comprises scrambled sequences of reference (R)-peptides that relate its abundance to a single protein standard (bovine serum albumin, BSA). FastCAT not only alleviates the need to purify CP or use sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) but also improves the accuracy, precision, and dynamic range of the absolute quantification by grouping Q-peptides according to the expected abundance of the target proteins. We benchmarked FastCAT against the reference method of MS Western and tested it in the direct molar quantification of neurological markers in human cerebrospinal fluid at the low ng/mL level.

A family of QconCATs (Quantification conCATemers) for the quantification of human pharmacological target proteins.

We have developed a family of QconCAT standards for the absolute quantification of pharmacological target proteins in a variety of human tissues. The QconCATs consist of concatenated proteotypic peptides, are designed in silico, and expressed in E. coli in media enriched with [13C6] arginine and [13C6] lysine to generate stable isotope-labeled multiplexed absolute quantification standards. The so-called MetCAT (used to quantify cytochrome P450 (CYP) and glucuronosyltransferase (UGT) enzymes), the liver TransCAT (used to quantify plasma-membrane drug transporters) and the brain TransCAT (used to quantify transporters expressed in the blood-brain barrier) were previously reported. We now report new QconCATs for the quantification of non-UGT non-CYP drug metabolizing enzymes (NuncCAT) and receptor tyrosine kinases (KinCAT). We have also redesigned the liver TransCAT, replacing problematic peptides and the N-terminal tag, for better characterization and expression. All these QconCATs showed high purity, high labelling efficiency with stable 13C isotope (>95%), and high sequence coverage (>88%). They represent a close-knit family of standards for quantifying pharmacokinetic targets, together with a more distant cousin, the KinCAT, used to quantify pharmacodynamic targets. SIGNIFICANCE: Multiplexed determination of absolute protein abundances using quantitative conCATemers (QconCATs) has already been successfully demonstrated in different human tissues. We have previously reported two QconCATs; MetCAT and TransCAT, for the quantification of key enzymes (cytochrome P450 enzymes (CYP) and glucuronosyltransferases (UGT)) and drug transporters. To build on these reports, application of the QconCAT methodology for the determination of non-UGT non-CYP enzymes and receptor tyrosine kinases (RTKs) in human tissue is reported here. This report focuses on development and characterization of two QconCAT constructs for the quantification of 24 enzymes and 21 RTKs. We demonstrate that the developed QconCATs have high purity, high incorporation efficiency and low peptide miscleavage upon proteolysis. Application of these QconCATs for reliable quantification of target proteins was achieved in human liver.

Proteomics of colorectal cancer liver metastasis: A quantitative focus on drug elimination and pharmacodynamics effects.

AIMS: This study aims to quantify drug-metabolising enzymes, transporters, receptor tyrosine kinases (RTKs) and protein markers (involved in pathways affected in cancer) in pooled healthy, histologically normal and matched cancerous liver microsomes from colorectal cancer liver metastasis (CRLM) patients. METHODS: Microsomal fractionation was performed and pooled microsomes were prepared. Global and accurate mass and retention time liquid chromatography-mass spectrometry proteomics were used to quantify proteins. A QconCAT (KinCAT) for the quantification of RTKs was designed and applied for the first time. Physiologically based pharmacokinetic (PBPK) simulations were performed to assess the contribution of altered abundance of drug-metabolising enzymes and transporters to changes in pharmacokinetics. RESULTS: Most CYPs and UGTs were downregulated in histologically normal relative to healthy samples, and were further reduced in cancer samples (up to 54-fold). The transporters, MRP2/3, OAT2/7 and OATP2B1/1B3/1B1 were downregulated in CRLM. Application of abundance data in PBPK models for substrates with different attributes indicated substantially lower (up to 13-fold) drug clearance when using cancer-specific instead of default parameters in cancer population. Liver function markers were downregulated, while inflammation proteins were upregulated (by up to 76-fold) in cancer samples. Various pharmacodynamics markers (e.g. RTKs) were altered in CRLM. Using global proteomics, we examined proteins in pathways relevant to cancer (such as metastasis and desmoplasia), including caveolins and collagen chains, and confirmed general over-expression of such pathways. CONCLUSION: This study highlights impaired drug metabolism, perturbed drug transport and altered abundance of cancer markers in CRLM, demonstrating the importance of population-specific abundance data in PBPK models for cancer.

Construction of à la carte QconCAT protein standards for multiplexed quantification of user-specified target proteins.

BACKGROUND: QconCATs are quantitative concatamers for proteomic applications that yield stoichiometric quantities of sets of stable isotope-labelled internal standards. However, changing a QconCAT design, for example, to replace poorly performing peptide standards has been a protracted process. RESULTS: We report a new approach to the assembly and construction of QconCATs, based on synthetic biology precepts of biobricks, making use of loop assembly to construct larger entities from individual biobricks. The basic building block (a Qbrick) is a segment of DNA that encodes two or more quantification peptides for a single protein, readily held in a repository as a library resource. These Qbricks are then assembled in a one tube ligation reaction that enforces the order of assembly, to yield short QconCATs that are useable for small quantification products. However, the DNA context of the short construct also allows a second cycle of loop assembly such that five different short QconCATs can be assembled into a longer QconCAT in a second, single tube ligation. From a library of Qbricks, a bespoke QconCAT can be assembled quickly and efficiently in a form suitable for expression and labelling in vivo or in vitro. CONCLUSIONS: We refer to this approach as the ALACAT strategy as it permits à la carte design of quantification standards. ALACAT methodology is a major gain in flexibility of QconCAT implementation as it supports rapid editing and improvement of QconCATs and permits, for example, substitution of one peptide by another.

Injectable Myocardial Matrix Hydrogel Mitigates Negative Left Ventricular Remodeling in a Chronic Myocardial Infarction Model.

A first-in-man clinical study on a myocardial-derived decellularized extracellular matrix hydrogel suggested the potential for efficacy in chronic myocardial infarction (MI) patients. However, little is understood about the mechanism of action in chronic MI. In this study, the authors investigated the efficacy and mechanism by which the myocardial matrix hydrogel can mitigate negative left ventricular (LV) remodeling in a rat chronic MI model. Assessment of cardiac function via magnetic resonance imaging demonstrated preservation of LV volumes and apical wall thickening. Differential gene expression analyses showed the matrix is able to prevent further negative LV remodeling in the chronic MI model through modulation of the immune response, down-regulation of pathways involved in heart failure progression and fibrosis, and up-regulation of genes important for cardiac muscle contraction.

Hybrid QconCAT-Based Targeted Absolute and Data-Independent Acquisition-Based Label-Free Quantification Enables In-Depth Proteomic Characterization of Wheat Amylase/Trypsin Inhibitor Extracts.

Wheat amylase/trypsin inhibitors (ATIs) have gained significant relevance as inducers of intestinal and extra-intestinal inflammation. In this study, we present a novel hybrid data-independent acquisition (DIA) liquid chromatography-mass spectrometry (LC-MS) approach, combining QconCAT technology with short microflow LC gradients and DIA and apply the method toward the quantitative proteome analysis of ATI extracts. The presented method is fast, robust, and reproducible and provides precise QconCAT-based absolute quantification of major ATI proteins while simultaneously quantifying the proteome by label-free quantification (LFQ). We analyzed extracts of 60 varieties of common wheat grown in replication and evaluated the reproducibility and precision of the workflow for the quantification of ATIs. Applying the method to analyze different wheat species (i.e., common wheat, spelt, durum wheat, emmer, and einkorn) and comparing the results to published data, we validated inter-laboratory and cross-methodology reproducibility of ATI quantification, which is essential in the context of large-scale breeding projects. Additionally, we applied our workflow to assess environmental effects on ATI expression, analyzing ATI content and proteome of same varieties grown at different locations. Finally, we explored the potential of combining QconCAT-based absolute quantification with DIA-based LFQ proteome analysis for the generation of new hypotheses or assay development.

Overexpression of Sedoheptulose-1,7-Bisphosphatase Enhances Photosynthesis in Chlamydomonas reinhardtii and Has No Effect on the Abundance of Other Calvin-Benson Cycle Enzymes.

The productivity of plants and microalgae needs to be increased to feed the growing world population and to promote the development of a low-carbon economy. This goal can be achieved by improving photosynthesis via genetic engineering. In this study, we have employed the Modular Cloning strategy to overexpress the Calvin-Benson cycle (CBC) enzyme sedoheptulose-1,7-bisphosphatase (SBP1) up to threefold in the unicellular green alga Chlamydomonas reinhardtii. The protein derived from the nuclear transgene represented ∼0.3% of total cell protein. Photosynthetic rate and growth were significantly increased in SBP1-overexpressing lines under high-light and elevated CO2 conditions. Absolute quantification of the abundance of all other CBC enzymes by the QconCAT approach revealed no consistent differences between SBP1-overexpressing lines and the recipient strain. This analysis also revealed that the 11 CBC enzymes represent 11.9% of total cell protein in Chlamydomonas. Here, the range of concentrations of CBC enzymes turned out to be much larger than estimated earlier, with a 128-fold difference between the most abundant CBC protein (rbcL) and the least abundant (triose phosphate isomerase). Accordingly, the concentrations of the CBC intermediates are often but not always higher than the binding site concentrations of the enzymes for which they act as substrates. The enzymes with highest substrate to binding site ratios might represent good candidates for overexpression in subsequent engineering steps.

A novel mass spectrometry method for the absolute quantification of several cytochrome P450 and uridine 5'-diphospho-glucuronosyltransferase enzymes in the human liver.

Cytochrome P450 (CYP450) and 5'-diphosphate glucuronosyltransferases (UGT) are the two major families of drug-metabolizing enzymes in the human liver microsome (HLM). As a result of their frequent abundance fluctuation among populations, the accurate quantification of these enzymes in different individuals is important for designing patient-specific dosage regimens in the framework of precision medicine. The preparation and quantification of internal standards is an essential step for the quantitative analysis of enzymes. However, the commonly employed stable isotope labeling-based strategy (QconCAT) suffers from requiring very expensive isotopic reagents, tedious experimental procedures, and long labeling times. Furthermore, arginine-to-proline conversion during metabolic isotopic labeling compromises the quantification accuracy. Therefore, we present a new strategy that replaces stable isotope-labeled amino acids with lanthanide labeling for the preparation and quantification of QconCAT internal standard peptides, which leads to a threefold reduction in the reagent costs and a fivefold reduction in the time consumed. The absolute amount of trypsin-digested QconCAT peptides can be obtained by lanthanide labeling and inductively coupled plasma-optical emission spectrometry (ICP-OES) analysis with a high quantification accuracy (%RE < 20%). By taking advantage of the highly selective and facile ICP-OES procedure and multiplexed large-scale absolute target protein quantification using biological mass spectrometry, this strategy was successfully used for the absolute quantification of drug-metabolizing enzymes. We obtained good linearity (correlation coefficient > 0.95) over concentrations spanning 2.5 orders of magnitude with improved sensitivity (limit of quantification = 2 fmol) in nine HLM samples, indicating the potential of this method for large-scale absolute target protein quantification in clinical samples. Graphical abstract.

PIKES Analysis Reveals Response to Degraders and Key Regulatory Mechanisms of the CRL4 Network.

Co-opting Cullin4 RING ubiquitin ligases (CRL4s) to inducibly degrade pathogenic proteins is emerging as a promising therapeutic strategy. Despite intense efforts to rationally design degrader molecules that co-opt CRL4s, much about the organization and regulation of these ligases remains elusive. Here, we establish protein interaction kinetics and estimation of stoichiometries (PIKES) analysis, a systematic proteomic profiling platform that integrates cellular engineering, affinity purification, chemical stabilization, and quantitative mass spectrometry to investigate the dynamics of interchangeable multiprotein complexes. Using PIKES, we show that ligase assemblies of Cullin4 with individual substrate receptors differ in abundance by up to 200-fold and that Cand1/2 act as substrate receptor exchange factors. Furthermore, degrader molecules can induce the assembly of their cognate CRL4, and higher expression of the associated substrate receptor enhances degrader potency. Beyond the CRL4 network, we show how PIKES can reveal systems level biochemistry for cellular protein networks important to drug development.

Characterization of CYP2B6 K262R allelic variants by quantitative allele-specific proteomics using a QconCAT standard.

Clinically-relevant proteins are routinely characterized by targeted proteomic methods, which offer high accuracy and reproducibility. However, assays developed for these techniques lack distinction between different alleles expressed in biological samples. The significance of assessing such variations in genes relevant to pharmacology will depend on their prevalence and effects on drug therapy. We propose quantitative allele-specific proteomics for simultaneous abundance measurement and determination of missense polymorphisms. We employed a targeted proteomic strategy using a QconCAT standard which included two surrogate peptides (at 1:1 ratio) for a prevalent variation of CYP2B6 (K262R) so that the two variants could be quantified directly. Measurement was carried out in 24 human liver samples, out of which 21 were genotyped. Allele-specific analysis of CYP2B6 expression was accurate and precise (CV < 9%), leading to determination of allele expression ratios (variant to wild type) for heterozygous (1.006 ±â€¯0.079, n = 12) and homozygous (0.005 ±â€¯0.004, n = 8) phenotypes. The abundance of CYP2B6 was 7.4 ±â€¯7.8 pmol mg-1 microsomal protein and showed good correlation with activity against mephenytoin (Rs = 0.91, p < 0.0001; R2 = 0.93). Comparable abundance (and activity) appeared to be associated with genotypes that express at least one wild type allele, which was corroborated by turnover values. This proof-of-principle study demonstrates the possibility of simultaneous determination of CYP2B6 phenotype and abundance by independent assessment of allele products.

Quantitative Proteomics of Enriched Esophageal and Gut Tissues from the Human Blood Fluke Schistosoma mansoni Pinpoints Secreted Proteins for Vaccine Development.

Schistosomes are blood-dwelling helminth parasites that cause schistosomiasis, a debilitating disease resulting in inflammation and, in extreme cases, multiple organ damage. Major challenges to control the transmission persist, and the discovery of protective antigens remains of critical importance for vaccine development. Rhesus macaques can self-cure following schistosome infection, generating antibodies that target proteins from the tegument, gut, and esophagus, the last of which is the least investigated. We developed a dissection technique that permitted increased sensitivity in a comparative proteomics profiling of schistosome esophagus and gut. Proteome analysis of the male schistosome esophagus identified 13 proteins encoded by microexon genes (MEGs), 11 of which were uniquely located in the esophageal glands. Based on this and transcriptome information, a QconCAT was designed for the absolute quantification of selected targets. MEGs 12, 4.2, and 4.1 and venom allergen-like protein 7 were the most abundant, spanning over 245 million to 6 million copies per cell, while aspartyl protease, palmitoyl thioesterase, and galactosyl transferase were present at <1 million copies. Antigenic variation by alternative splicing of MEG proteins was confirmed together with a specialized machinery for protein glycosylation/secretion in the esophagus. Moreover, some gastrodermal secretions were highly enriched in the gut, while others were more uniformly distributed throughout the parasite, potentially indicating lysosomal activity. Collectively, our findings provide a more rational, better-oriented selection of schistosome vaccine candidates in the context of a proven model of protective immunity.

Quantitative assay of targeted proteome in tomato trichome glandular cells using a large-scale selected reaction monitoring strategy.

BACKGROUND: Glandular trichomes found in vascular plants are called natural cell factories because they synthesize and store secondary metabolites in glandular cells. To systematically understand the metabolic processes in glandular cells, it is indispensable to analyze cellular proteome dynamics. The conventional proteomics methods based on mass spectrometry have enabled large-scale protein analysis, but require a large number of trichome samples for in-depth analysis and are not suitable for rapid and sensitive quantification of targeted proteins. RESULTS: Here, we present a high-throughput strategy for quantifying targeted proteins in specific trichome glandular cells, using selected reaction monitoring (SRM) assays. The SRM assay platform, targeting proteins in type VI trichome gland cells of tomato as a model system, demonstrated its effectiveness in quantifying multiple proteins from a limited amount of sample. The large-scale SRM assay uses a triple quadrupole mass spectrometer connected online to a nanoflow liquid chromatograph, which accurately measured the expression levels of 221 targeted proteins contained in the glandular cell sample recovered from 100 glandular trichomes within 120 min. Comparative quantitative proteomics using SRM assays of type VI trichome gland cells between different organs (leaves, green fruits, and calyx) revealed specific organ-enriched proteins. CONCLUSIONS: We present a targeted proteomics approach using the established SRM assays which enables quantification of proteins of interest with minimum sampling effort. The remarkable success of the SRM assay and its simple experimental workflow will increase proteomics research in glandular trichomes.

Functional Insights from the Proteomic Inventory of Ovine Forestomach Matrix.

Ovine forestomach matrix (OFM) is a decellularized extracellular matrix (dECM) biomaterial that serves as a scaffold for remodeling damaged soft tissue. dECM biomaterials are used in a variety of clinical applications, and their regenerative capacity is encoded not only in their biophysical properties but also in their molecular diversity. In this study, the proteome of OFM was characterized via both targeted and global mass spectrometry (MS) with the use of heavy isotope labeled (SIL) internal standards. Proteins were identified following either chemical digestion or extraction using saline or guanidine hydrochloride, followed by high resolution size exclusion chromatography. Identified proteins were annotated using the matrisome database and molecular function using the gene ontology database. The characterization identified 153 unique matrisome proteins, including 25 collagens, 58 glycoproteins, 12 proteoglycans, 13 ECM affiliated proteins, 20 ECM regulators, and 23 secreted factors. This inventory represents a comprehensive array of matrix proteins that are retained in OFM after processing. The diversity of proteins identified may contribute to OFM's remodeling capacity in clinical applications.

Functional Study of Carboxylesterase 1 Protein Isoforms.

Carboxylesterase 1 (CES1) is a primary human hepatic hydrolase involved in hydrolytic biotransformation of numerous medications. Considerable interindividual variability in CES1 expression and activity has been consistently reported. Four isoforms of the CES1 protein are produced by alternative splicing (AS). In the current study, the activity and expression of each CES1 isoform are examined using transfected cell lines, and CES1 isoform composition and its impact on CES1 activity in human livers are determined. In transfected cells, isoforms 3 and 4 show mRNA and protein expressions comparable to isoforms 1 and 2, but have significantly impaired activity when hydrolyzing enalapril and clopidogrel. In individual human liver samples, isoforms 1 and 2 are the major forms, contributing 73-90% of the total CES1 protein expression. In addition, the protein expression ratios of isoforms 1 and 2 to isoforms 3 and 4 are positively associated with CES1 activity in the liver, suggesting that CES1 isoform composition is a factor contributing to the variability in hepatic CES1 function. Further investigations of the regulation of CES1 AS would improve the understanding of CES1 variability and help develop a strategy to optimize the pharmacotherapy of many CES1 substrate medications.

Determining Allele-Specific Protein Expression (ASPE) Using a Novel Quantitative Concatamer Based Proteomics Method.

Measuring allele-specific expression (ASE) is a powerful approach for identifying cis-regulatory genetic variants. Here, we developed a novel targeted proteomics method for the quantification of allele-specific protein expression (ASPE) based on scheduled parallel reaction monitoring (PRM) with a heavy stable isotope-labeled quantitative concatamer (QconCAT) internal protein standard. This strategy was applied to the determination of the ASPE of UGT2B15 in human livers using the common UGT2B15 nonsynonymous variant rs1902023 (i.e., Y85D) as the marker to differentiate expressions from the two alleles. The QconCAT standard contains both the wild-type tryptic peptide and the Y85D mutant peptide at a ratio of 1:1 to ensure accurate measurement of the ASPE of UGT2B15. The results from 18 UGT2B15 Y85D heterozygotes revealed that the ratios between the wild-type Y allele and the mutant D allele varied from 0.60 to 1.46, indicating the presence of cis-regulatory variants. In addition, we observed no significant correlations between the ASPE and mRNA ASE of UGT2B15, suggesting the involvement of different cis-acting variants in regulating the transcription and translation processes of the gene. This novel ASPE approach provides a powerful tool for capturing cis-genetic variants involved in post-transcription processes, an important yet understudied area of research.

Identification and quantification of blood-brain barrier transporters in isolated rat brain microvessels.

The blood-brain barrier (BBB) maintains brain homeostasis by tightly regulating the exchange of molecules with systemic circulation. It consists primarily of microvascular endothelial cells surrounded by astrocytic endfeet, pericytes, and microglia. Understanding the make-up of transporters in rat BBB is essential to the translation of pharmacological and toxicological observations into humans. In this study, experimental workflows are presented in which the optimization of (a) isolation of rat brain microvessels (b) enrichment of endothelial cells, and (c) extraction and digestion of proteins were evaluated, followed by identification and quantification of BBB proteins. Optimization of microvessel isolation was indicated by 15-fold enrichment of endothelial cell marker Glut1 mRNA, whereas markers for other cell types were not enriched. Filter-aided sample preparation was shown to be superior to in-solution sample preparation (10251 peptides vs. 7533 peptides). Label-free proteomics was used to identify nearly 2000 proteins and quantify 1276 proteins in isolated microvessels. A combination of targeted and global proteomics was adopted to measure protein abundance of 6 ATP-binding cassette and 27 solute carrier transporters. Data analysis using proprietary Progenesis and open access MaxQuant software showed overall agreement; however, Abcb9 and Slc22a8 were quantified only by MaxQuant, whereas Abcc9 and Abcd3 were quantified only by Progenesis. Agreement between targeted and untargeted quantification was demonstrated for Abcb1 (19.7 ± 1.4 vs. 17.8 ± 2.3) and Abcc4 (2.2 ± 0.7 vs. 2.1 ± 0.4), respectively. Rigorous quantification of BBB proteins, as reported in this study, should assist with translational modeling efforts involving brain disposition of xenobiotics.

Stoichiometry, Absolute Abundance, and Localization of Proteins in the Bacillus cereus Spore Coat Insoluble Fraction Determined Using a QconCAT Approach.

Spores of Bacillus cereus pose a threat to food safety due to their high resistance to the heat or acid treatments commonly used to make food microbiologically safe. Spores may survive these treatments and later resume growth either on foodstuffs or, after ingestion, upon entering the gut they are capable of producing toxins, which cause either vomiting or diarrhea. The outer layers of the spore, the spore coat and exosporium, consist primarily of proteins that may serve as potential biomarkers for detection. The major morphogenetic protein CotE is important for correct assembly and attachment of the outermost layer, the exosporium, and by extension retention of many proteins. However, characterization of the proteins affected by deletion of CotE has been limited to electrophoretic patterns. Here we report the effect of CotE deletion on the insoluble fraction of the spore proteome through liquid chromatography-Fourier transform tandem mass spectrometry (LC-FTMS/MS) analysis. A total of 560 proteins have been identified in both mutant and wild-type spore coat isolates. A further 163 proteins were identified exclusively in wild-type spore isolates indicating that they are dependent on CotE for their association with the spore. Several of these are newly confirmed as associated with the exosporium, namely BC_2569 (BclF), BC_3345, BC_2427, BC_2878, BC_0666, BC_2984, BC_3481, and BC_2570. A total of 153 proteins were only identified in ΔCotE spore isolates. This was observed for proteins that are known or likely to be interacting with or are encased by CotE. Crucial spore proteins were quantified using a QconCAT reference standard, the first time this was used in a biochemically heterogeneous system. This allowed us to determine the absolute abundance of 21 proteins, which spanned across three orders of magnitude and together covered 5.66% ± 0.51 of the total spore weight. Applying the QconCAT methodology to the ΔCotE mutant allowed us to quantify 4.13% ± 0.14 of the spore total weight and revealed a reduction in abundance for most known exosporium associated proteins upon CotE deletion. In contrast, several proteins, either known or likely to be interacting with or encased by CotE (i.e., GerQ), were more abundant. The results obtained provide deeper insight into the layered spore structure such as which proteins are exposed on the outside of the spore. This information is important for developing detection methods for targeting spores in a food safety setting. Furthermore, protein stoichiometry and determination of the abundance of germination mediating enzymes provides useful information for germination and outgrowth model development.

Sample Preparation for Mass-Spectrometry Based Absolute Protein Quantification in Antibiotic Stress Research.

Absolute protein quantification is an essential tool for system biology approaches and elucidation of stoichiometry of multi-protein complexes. In this chapter, a universal protocol for gel free absolute protein quantification in bacterial systems is described, which can be used for sample preparation prior to miscellaneous mass-spectrometry-based quantification workflows like AQUA, Hi3, and emPAI. In addition, a focus has been set to the specific challenges in antibiotic stress research.