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Extracellular vesicles (EVs) represent a universal mechanism of intercellular communication in normal and pathological conditions. There are reports showing the presence of complement proteins in EV preparations, specifically those that can form a membrane attack complex (MAC). In the present work, we have used a quantitative mass spectrometry method that allows for the measurement of multiple targeted proteins in one experimental run. The quantification of MAC-forming proteins, namely C5b, C6, C7, C8, and C9, in highly purified EVs from normal human plasma revealed the presence of MAC proteins at approximately equal stoichiometry that does not fit the expected stoichiometry of preformed MAC. We concluded that while MAC proteins can be associated with EVs from normal plasma and presumably can be delivered to the recipient cells, there is no evidence that the EVs carry preformed MAC.

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Mitochondrial content has been reported outside of cells either within extracellular vesicles (EVs) or as free mitochondria. Mitochondrial EVs can potentially play multiple physiological and pathophysiological roles. To understand their functions, isolation protocols to separate mitochondrial EVs from other mitochondrial content need to be established. In the present work, we use a multiple reaction monitoring assay with isotope labeled internal standards to quantify 11 mitochondrial, 6 plasma membrane-specific, 4 endosomal membrane-specific, and 2 soluble proteins to evaluate the efficiency of chromatographic isolation of mitochondrial EVs. The isolation protocol includes ultracentrifugation, size exclusion chromatography, and chromatography on immobilized heparin. All protein concentrations were normalized to the concentration of ATP synthase alpha subunit to generate a ratio that allows comparison of different samples obtained during the isolation. We have shown that initial samples after ultracentrifugation are contaminated with non-EV mitochondrial content that cannot be separated from EVs using size exclusion chromatography, but can be efficiently separated from EVs on the column with immobilized heparin.

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Absolute quantification with mass spectrometry and isotope labeled internal standards has found broad applications in biomedical research. In the present research, it was used for developing and evaluating a new affinity-based approach to isolate extracellular vesicles (EVs) from human plasma. First, a phage display peptide library was screened against EVs as a bait and absolute quantification of multiple proteins helped to select the best bait available. Then, absolute quantification was used to evaluate the efficiency of affinity chromatography on peptide-Sepharose. In summary, we have demonstrated that peptides with affinity to EVs selected from phage library screening can be valuable ligands for EVs isolation. SIGNIFICANCE: Extracellular vesicles (EVs) have an important role in intercellular communication for all cell types. This makes EVs a promising new type of therapeutics capable to deliver drugs to specific sites with no off-target side effects. However, their isolation, and correct assignment of their biological function and properties remains an obscure field of research. In this study, we proposed to use MRM quantitation of a pattern of EVs and non-EVs proteins to develop a purification protocol based on affinity peptides selected from phage library screening. MRM quantification of EVs proteins can also help in identifying those that are subpopulation specific markers for further target-specific isolation.

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Extracellular vesicles (EVs) are traditionally divided into two major groups: (i) large vesicles originating from plasma membrane and called microvesicles, and (ii) small vesicles originating from the endoplasmic membrane and called exosomes. However, it is increasingly clear that the actual composition of a particular EV preparation cannot be adequately described with these two simple terms and is much more complex. Since the cell membrane origin of EVs predetermines their biological functions, the understanding of EV biogenesis is important for accurate interpretation of observed results. In the present study, we propose to take advantage of selective expression of some proteins in plasma or endosomal membranes and to use these proteins as plasma membrane-specific or endosomal membrane-specific markers. We have demonstrated that a quantitative mass spectrometry analysis allows simultaneous measurement of plasma membrane-specific and endosomal membrane-specific proteins in microvesicles and exosomes obtained after differential ultracentrifugation. Before mass spectrometry analysis, we also used sonicated platelets as a model of mixed EVs and multidetector asymmetrical-flow field-flow fractionation as an analytical method to verify a possible cross contamination of obtained microvesicles and exosomes. Based on the quantitative appearance of membrane-specific protein markers in EV preparations from human plasma and from human ARPE-19 cell medium, we concluded that there is no actual size limitation and both microvesicles and exosomes can be represented by large and small vesicles.

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A proof of concept for new methodology to detect and potentially quantify mAb aggregation is presented. Assay development included using an aggregated mAb as bait for screening of a phage display peptide library and identifying those peptides with random sequence which can recognize mAb aggregates. The selected peptides can be used for developing homogeneous quantitative methods to assess mAb aggregation. Results indicate that a peptide-binding method coupled with fluorescence polarization detection can detect mAb aggregation and potentially monitor the propensity of therapeutic protein candidates to aggregate.

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In the last decade, filamentous M13 bacteriophage has emerged into numerous biotechnological applications as a promising nontoxic and self-assembling biomaterial with specific binding properties. This raises a question about its upscale production that consequently requires an accurate phage enumeration during the various protocol developments. However, traditional methods of measuring phage concentration are mainly biological in nature and therefore time and labor intensive. These traditional methods also demonstrate poor reproducibility and are semi-quantitative at best. In the present work, we capitalized on mass spectrometry based absolute protein quantitation. We have optimized the quantitation conditions for a major coat protein, pVIII. Enumeration of M13 bacteriophage can be further performed using the determined molar concentration of pVIII, Avogadro's number, and known copy number of pVIII per phage. Since many different phages have well-defined copy number of capsid proteins, the proposed approach can be simply applied to any phage with known copy number of a specific capsid protein.

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Circulating in blood, extracellular vesicles (EVs) and lipoprotein particles (LPs) have diagnostic and prognostic value. To unambiguously define their functions, separation protocols need to be developed. However, because of their similar size and density, traditional approaches to separate EVs and LPs often fail to provide the required resolution. Further development and standardization of affinity-based protocols is necessary, and a quantitative method is needed to assess the efficiency of LP depletion from EV samples. In the present study, we propose the simultaneous quantification of three groups of proteins by mass spectrometry as a toolbox to evaluate prospective separation protocols. We generated 15N-labeled internal standards for quantification of (i) EV-specific proteins, (ii) all classes and subclasses of apolipoproteins constituting LPs, and (iii) several major serum proteins. These standards were then used in multiple reaction monitoring assay to evaluate the performance of size-exclusion chromatography, heparin-Sepharose, lipopolysaccharide-Sepharose, (2-hydroxypropyl)-ß-cyclodextrin-Sepharose, and concanavalin A-Sepharose in separating serum EVs and LPs. The efficiency of a resin to separate EVs from non-EV substances could be jeopardized by simultaneous EV aggregation. Therefore, dynamic light scattering analysis was used in this study in addition to the proteomic toolbox when making a recommendation to use particular resin for EV isolation. On the basis of our measurements, we concluded that none of the individual separation protocols used in this study resulted in LP-free EVs, and the combination of two protocols may be complex due to low EV yield. Overall, this further points to the importance of proposed proteomic toolbox for the future evaluation of EV separation protocols.

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The increasing interest in extracellular vesicles (EVs) research is fueled by reports indicating their unique role in intercellular communication and potential connection to the development of common human diseases. The unique role assumes unique protein and nucleic acid cargo. Unfortunately, accurate analysis of EVs cargo faces a challenge of EVs isolation. Generally used isolation techniques do not separate different subtypes of EVs and even more, poorly separate EVs from non-EVs contaminants. Further development of EVs isolation protocols urgently needs a quantitative method of EVs purity assessment. We report here that multiple reaction monitoring assay using internal standards carrying peptides for quantification of EVs and non-EVs proteins is a suitable approach to assess purity of EVs preparations. As a first step in potential standardization of EVs isolation, we have evaluated polymer-based precipitation techniques and compared them to traditional ultracentrifugation protocol.

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Cytochrome P450 27A1 (CYP27A1 or sterol 27-hydroxylase) is a ubiquitous, multifunctional enzyme catalyzing regio- and stereospecific hydroxylation of different sterols. In humans, complete CYP27A1 deficiency leads to cerebrotendinous xanthomatosis or nodule formation in tendons and brain (preferentially in the cerebellum) rich in cholesterol and cholestanol, the 5α-saturated analog of cholesterol. In Cyp27a1-/- mice, xanthomas are not formed, despite a significant cholestanol increase in the brain and cerebellum. The mechanism behind cholestanol production has been clarified, yet little is known about its metabolism, except that CYP27A1 might metabolize cholestanol. It also is unclear why CYP27A1 deficiency results in preferential cholestanol accumulation in the cerebellum. We hypothesized that cholestanol might be metabolized by CYP46A1, the principal cholesterol 24-hydroxylase in the brain. We quantified sterols along with CYP27A1 and CYP46A1 in mouse models (Cyp27a1-/-, Cyp46a1-/-, Cyp27a1-/-Cyp46a1-/-, and two wild type strains) and human brain specimens. In vitro experiments with purified P450s were conducted as well. We demonstrate that CYP46A1 is involved in cholestanol removal from the brain and that several factors contribute to the preferential increase in cholestanol in the cerebellum arising from CYP27A1 deficiency. These factors include (i) low cerebellar abundance of CYP46A1 and high cerebellar abundance of CYP27A1, the lack of which probably selectively increases the cerebellar cholestanol production; (ii) spatial separation in the cerebellum of cholesterol/cholestanol-metabolizing P450s from a pool of metabolically available cholestanol; and (iii) weak cerebellar regulation of cholesterol biosynthesis. We identified a new physiological role of CYP46A1, an important brain enzyme and cytochrome P450 that could be activated pharmacologically.

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Using mAbs as therapeutic molecules is complicated by the propensity of mAbs to aggregate at elevated concentrations, which can lead to a variety of adverse events in treatment. Here, we describe a proof-of-concept for new methodology to detect and quantify mAb aggregation. Assay development included using an aggregated mAb as bait for screening of phage display peptide library and identifying those peptides with random sequence which can recognize mAb aggregates. Once identified, the selected peptides can be used for developing quantitative methods to assess mAb aggregation. Results indicate that a peptide binding method coupled with mass spectrometric detection of bound peptide can quantify mAb aggregation and potentially be useful for monitoring aggregation propensity of therapeutic protein candidates.

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Cytochrome P450 46A1 (CYP46A1) is a microsomal enzyme and cholesterol 24-hydroxylase that controls cholesterol elimination from the brain. This P450 is also a potential target for Alzheimer disease because it can be activated pharmacologically by some marketed drugs, as exemplified by efavirenz, the anti-HIV medication. Previously, we suggested that pharmaceuticals activate CYP46A1 allosterically through binding to a site on the cytosolic protein surface, which is different from the enzyme active site facing the membrane. Here we identified this allosteric site for efavirenz on CYP46A1 by using a combination of hydrogen-deuterium exchange coupled to MS, computational modeling, site-directed mutagenesis, and analysis of the CYP46A1 crystal structure. We also mapped the binding region for the CYP46A1 redox partner oxidoreductase and found that the allosteric and redox partner binding sites share a common border. On the basis of the data obtained, we propose the mechanism of CYP46A1 allostery and the pathway for the signal transmission from the P450 allosteric site to the active site.

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Protein quantification based on stable isotope labeling-mass spectrometry involves adding known quantities of stable isotope-labeled internal standards into biological samples. The internal standards are analogous to analyte molecules and quantification is achieved by comparing signals from isotope-labeled and analyte molecules. This methodology is broadly applicable to proteomics research, biomarker discovery and validation, and clinical studies, which require accurate and precise protein abundance measurements. One such internal standard platform for protein quantification is concatenated peptides (QconCAT). This chapter describes a protocol for the design, expression, characterization, and application of the QconCAT strategy for protein quantification.

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Glycan is an important class of macromolecules that play numerous biological functions. Quantitative glycomics--analysis of glycans at global level--however, is far behind genomics and proteomics owing to technical challenges associated with their chemical properties and structural complexity. As a result, technologies that can facilitate global glycan analysis are highly sought after. Here, we present QUANTITY (Quaternary Amine Containing Isobaric Tag for Glycan), a quantitative approach that can not only enhance detection of glycans by mass spectrometry, but also allow high-throughput glycomic analysis from multiple biological samples. This robust tool enabled us to accomplish glycomic survey of bioengineered Chinese Hamster Ovary (CHO) cells with knock-in/out enzymes involved in protein glycosylation. Our results demonstrated QUANTITY is an invaluable technique for glycan analysis and bioengineering.

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The Borrelia burgdorferi spirochete is the causative agent of Lyme disease, the most common tick-borne disease in the United States. The low abundance of bacterial proteins in human serum during infection imposes a challenge for early proteomic detection of Lyme disease. To address this challenge, we propose to detect membrane proteins released from bacteria due to disruption of their plasma membrane triggered by the innate immune system. These membrane proteins can be separated from the bulk of serum proteins by high-speed centrifugation causing substantial sample enrichment prior to targeted protein quantification using multiple reaction monitoring mass spectrometry. This new approach was first applied to detection of B. burgdorferi membrane proteins supplemented in human serum. Our results indicated that detection of B. burgdorferi membrane proteins, which are ≈10(7) lower in abundance than major serum proteins, is feasible. Therefore, quantitative analysis was also carried out for serum samples from three patients with acute Lyme disease. We were able to demonstrate the detection of ospA, the major B. burgdorferi lipoprotein at the level of 4.0 fmol of ospA/mg of serum protein. The results confirm the concept and suggest that the proposed approach can be expanded to detect other bacterial infections in humans, particularly where existing diagnostics are unreliable.

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BACKGROUND: Alzheimer's disease (AD) is the sixth leading cause of death and the most costly disease in the US. Despite the enormous impact of AD, there are no treatments that delay onset or stop disease progression currently on the market. This is partly due to the complexity of the disease and the largely unknown pathogenesis of sporadic AD, which accounts for the vast majority of cases. Epigenetics has been implicated as a critical component to AD pathology and a potential "hot spot" for treatments. Histone post-translational modifications (PTMs) are a key element in epigenetic regulation of gene expression and are known to be associated with the pathology of numerous diseases. Investigation of histone PTMs can help elucidate AD pathology and identify targets for therapies. RESULTS: A multiple reaction monitoring mass spectrometry assay was used to measure changes in abundance of several histone PTMs in frontal cortex from human donors affected with AD (n = 6) and age-matched, normal donors (n = 6). Of the changes observed, notable decreases in methylation of H2B residue K108 by 25 % and H4 residue R55 by 35 % were measured and are likely associated with hydrogen bonding networks important for nucleosome stability. Additionally, a 91 % increase in ubiquitination of K120 on H2B was measured as well as an apparent loss in acetylation of the region near the N-terminus of H4. Our method of quantification was also determined to be precise and robust, signifying measured changes were representative of true biological differences between donors and sample groups. CONCLUSION: We are the first to report changes in methylation of H2B K108, methylation of H4 R55, and ubiquitination of H2B K120 in frontal cortex from human donors with AD. These notable PTM changes may be of great importance in elucidating the epigenetic mechanism of AD as it relates to disease pathology. Beyond the structural and functional impacts of the changes we have measured, the sites of altered PTMs may be used to identify enzymes responsible for their modulation, which could be used as prospective drug targets for highly specific AD therapies.

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Alzheimer's disease has been shown to have a global reduction in gene expression, called an epigenetic blockade, which may be regulated by histone post-translational modifications. Histone H3 has been shown to be highly regulated by phosphorylation. We, therefore, chose H3 for investigation of phosphorylation of the core sites serine-57 (S57) and threonine-58 (T58). Hemispheres of brains from a mouse model of rapid amyloid deposition (5XFAD) were used for measurement of S57 and T58 phosphorylation. Multiple reaction monitoring (MRM) was used to measure the level of phosphorylation, which was normalized to a non-modified "housekeeping" peptide of H3. S57 phosphorylation was decreased by 40%, T58 phosphorylation was decreased by 45%, and doubly phosphorylated S57pT58p was decreased by 30% in 5XFAD brain in comparison to C57BL/6J age- and sex-matched wild type controls. Amyloid-ß (Aß) and amyloid precursor protein were also measured to confirm that 5XFAD mice produced high levels of Aß. Decreased phosphorylation of these sites in close proximity to DNA may lead to stabilization of DNA-histone interactions and a condensed chromatin state, consistent with the epigenetic blockade associated with AD. Our findings of H3 sites S57 and T58 exhibiting lower levels of phosphorylation in 5XFAD model compared to wild type control implicate these sites in the epigenetic blockade in neurodegeneration pathology.

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Histone deacetylase (HDAC) inhibition has promise as a therapy for Alzheimer's disease (AD) and other neurodegenerative diseases. Currently, therapeutic HDAC inhibitors target many HDAC isoforms, a particularly detrimental approach when HDAC isoforms are known to have different and specialized functions. We have developed a multiple reaction monitoring (MRM) mass spectrometry assay using stable isotope-labeled QconCATs as internal standards to quantify HDAC isoforms. We further determined a quantitative pattern of specific HDACs expressed in various human and mouse neural tissues. In human AD frontal cortex, HDAC1,2 decreased 32%, HDAC5 increased 47%, and HDAC6 increased 31% in comparison to age-matched controls. Human neural retina concentrations of HDAC1, 2, HDAC5, HDAC6, and HDAC7 decreased in age-related macular degeneration (AMD)-affected donors and exhibited a greater decrease in AD-affected donors in comparison to age-matched control neural retinas. Additionally, HDAC concentrations were measured in whole hemisphere of brain of 5XFAD mice, a model of ß-amyloid deposition, to assess similarity to AD in human frontal cortex. HDAC profiles of human frontal cortex and mouse hemisphere had noticeable differences and relatively high concentrations of HDAC3 and HDAC4 in mice, which were undetectable in humans. Our method for quantification of HDAC isoforms is a practical and efficient technique to quantify isoforms in various tissues and diseases. Changes in HDAC concentrations reported herein contribute to the understanding of the pathology of neurodegeneration.

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Stable-isotope-labeling mass spectrometry involves the addition of known quantities of stable-isotope labeled standards, which mimic native molecules, to biological samples. We evaluated three conventional internal standard platforms (synthetic peptides, QconCAT constructs, and recombinant proteins) for quantitative accuracy, precision, and inherent advantages and limitations. Internal standards for the absolute quantification of three human cytokine proteins (interferon gamma, interleukin-1 beta, and tumor necrosis factor alpha) were designed and verified. Multiple reaction monitoring assays, calibration curve construction, and regression analysis were used to assess quantitative performance of the internal standard platforms. We also investigated a strategy for methodological improvement to current platforms using natural flanking sequences. Data analysis revealed that full length protein standards have the broadest quantitative reliability with accuracy being peptide-dependent for QconCATs and synthetic peptides. Natural flanking sequences greatly improved the quantitative performance of both QconCAT and synthetic peptide standards.

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Quantification by targeted proteomics has largely depended on mass spectrometry and isotope-labeled internal standards. In addition to traditionally used recombinant proteins or synthetic peptides, concatenated peptides (QconCATs) were introduced as a conceptually new source of internal standard. In the present study, we focused on assessing the length of natural flanking sequences, which surround each peptide included in QconCAT and provide for identical rates of analyte and standard digestion by trypsin. We have expressed, purified, and characterized a set of seven (15)N-labeled QconCATs that cover seven tryptic peptides from human clusterin with a length of natural flanking sequences ranging from none (+0) to six amino acid residues (+6) for each tryptic peptide. Individual QconCATs were mixed with recombinant human clusterin at a 1:1 molar ratio and digested, and the actual ratios for each combination of peptide/flanking sequence were measured with a multiple reaction monitoring assay. Data analysis suggested that natural flanking sequences shorter than +6 residues can cause a quantitative error because the random appearance of other amino acid residues in close proximity to trypsin cleavage sites has unpredictable consequences for the digestion rates of QconCATs.

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Carbamate kinase from Giardia lamblia is an essential enzyme for the survival of the organism. The enzyme catalyzes the final step in the arginine dihydrolase pathway converting ADP and carbamoyl phosphate to ATP and carbamate. We previously reported that disulfiram, a drug used to treat chronic alcoholism, inhibits G. lamblia CK and kills G. lamblia trophozoites in vitro at submicromolar IC50 values. Here, we examine the structural basis for G. lamblia CK inhibition of disulfiram and its analog, thiram, their activities against both metronidazole-susceptible and metronidazole-resistant G. lamblia isolates, and their efficacy in a mouse model of giardiasis. The crystal structure of G. lamblia CK soaked with disulfiram revealed that the compound thiocarbamoylated Cys-242, a residue located at the edge of the active site. The modified Cys-242 prevents a conformational transition of a loop adjacent to the ADP/ATP binding site, which is required for the stacking of Tyr-245 side chain against the adenine moiety, an interaction seen in the structure of G. lamblia CK in complex with AMP-PNP. Mass spectrometry coupled with trypsin digestion confirmed the selective covalent thiocarbamoylation of Cys-242 in solution. The Giardia viability studies in the metronidazole-resistant strain and the G. lamblia CK irreversible inactivation mechanism show that the thiuram compounds can circumvent the resistance mechanism that renders metronidazole ineffectiveness in drug resistance cases of giardiasis. Together, the studies suggest that G. lamblia CK is an attractive drug target for development of novel antigiardial therapies and that disulfiram, an FDA-approved drug, is a promising candidate for drug repurposing.

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