Sequential Windowed Acquisition Of All Theoretical Fragment Ion Mass Spectra Research Articles

Proteomic response of Pseudomonas aeruginosa IIPIS-8 during rapid and efficient degradation of naphthalene

Polycyclic aromatic hydrocarbons (PAHs) are widely distributed in the ecosystem and are of significant concern due to their toxicity and mutagenicity. Bioremediation of PAHs is a popular and benign approach that ameliorates the environment. This study investigated the biodegradation and proteome response of Pseudomonas aeruginosa IIPIS-8 for two-ringed PAH: naphthalene (NAP) to understand proteome alteration during its bioremediation. Rapid biodegradation was observed up to 98 ± 1.26% and 84 ± 1.03%, respectively, for initial concentrations of 100 mg L−1 and 500 mg L−1 of NAP. Degradation followed first-order kinetics with rate constants of 0.12 h−1 and 0.06 h−1 and half-life (t1/2) of 5.7 h and 11.3 h, respectively. Additionally, the occurrence of key ring cleavage and linear chain intermediates, 2,3,4,5,6, -pentamethyl acetophenone, 1-octanol 2-butyl, and hexadecanoic acid supported complete NAP degradation. Proteomics study of IIPIS-8 throws light on the impact of protein expression, in which 415 proteins were quantified in sequential windowed acquisition of all theoretical fragment ion mass spectra (SWATH-MS) analysis, of which 97 were found to be significantly up-regulated and 75 were significantly down-regulated by ≥ 2-fold change (p values ≤ 0.05), during the NAP degradation. The study also listed the up-regulation of several enzymes, including oxido-reductases, hydrolases, and catalases, potentially involved in NAP degradation. Overall, differential protein expression, through proteomics study, demonstrated IIPIS-8's capability to efficiently assimilate NAP in their metabolic pathways even in a high concentration of NAP.

Comparison of tropomyosin released peptide and epitope mapping after in vitro digestion from fish (Larimichthys crocea), shrimp (Litopenaeus vannamei) and clam (Ruditapes philippinarum) through SWATH-MS based proteomics

Tropomyosin (TM) is a major shellfish allergen and a minor fish allergen. Different digestion profiles affect potential allergen anaphylaxis of protein. In this study, released peptides of fish-TM, shrimp-TM, and clam-TM by in vitro digestion of simulated gastric fluid (SGF), simulated intestinal fluid (SIF), and gastrointestinal (GI) were analyzed using sequential windowed acquisition of all theoretical fragment ion mass spectra (SWATH-MS) based proteomics. Results showed that digestion products of shrimp-TM yielded a lot of peptides matched T/B cell epitopes while core regions matched epitopes were distributed along the entire chain. Pepsin or trypsin-based digestion products of shrimp-TM presented many more peptides matched T/B cell epitopes compared with those of fish-TM and clam-TM. Besides, a differentiating peptide of VEKDKALSNAEGEVAAL (72–88) overlapped T/B cell epitopes could be used as a candidate peptide marker to identify tropomyosin allergen. These findings would supply new insight into the different allergenicity of tropomyosin.

Decoding the radiomic and proteomic phenotype of epicardial adipose tissue associated with adverse left atrial remodelling and post-operative atrial fibrillation in aortic stenosis

Epicardial adipose tissue (EAT) volume and attenuation on computed tomography (CT) have been associated with atrial fibrillation. Beyond these conventional CT measures, radiomics allows extraction of high-dimensional data and deep quantitative adipose tissue phenotyping, which may capture its underlying biology. We aimed to explore the EAT proteomic and CT-radiomic signatures associated with impaired left atrial (LA) remodelling and post-operative atrial fibrillation (POAF). We prospectively included 132 patients with severe aortic stenosis with no prior atrial fibrillation referred for aortic valve replacement. Pre-operative non-contrast CT images were obtained for extraction of EAT volume and other radiomic features describing EAT texture. The LA function was assessed by 2D-speckle-tracking echocardiography peak atrial longitudinal strain and peak atrial contraction strain. The EAT biopsies were performed during surgery for proteomic analysis by sequential windowed acquisition of all theoretical fragment ion mass spectra (SWATH-MS). The POAF incidence was monitored from surgery until discharge. Impaired LA function and incident POAF were associated with EAT up-regulation of inflammatory and thrombotic proteins, and down-regulation of cardioprotective proteins with anti-inflammatory and anti-lipotoxic properties. The EAT volume was independently associated with LA enlargement, impaired function, and POAF risk. On CT images, EAT texture of patients with POAF was heterogeneous and exhibited higher maximum grey-level values than sinus rhythm patients, which correlated with up-regulation of inflammatory and down-regulation of lipid droplet-formation EAT proteins. The CT radiomics of EAT provided an area under the curve of 0.80 (95% confidence interval: 0.68-0.92) for discrimination between patients with POAF and sinus rhythm. Pre-operative CT-radiomic profile of EAT detected adverse EAT proteomics and identified patients at risk of developing POAF.

SWATH-MS-based proteomics reveals functional biomarkers of Th1/Th2 responses of tropomyosin allergy in mouse models

Type-I food allergies are hypersensitive reactions compromising the immune organs and epithelial barriers. To investigate the organ-specific proteomic alterations of the allergy responses, the spleen and intestine of mice sensitized with high (shrimp and clam) and weak (fish) allergenic tropomyosins were analyzed using sequential windowed acquisition of all theoretical fragment ion mass spectra (SWATH-MS)-based proteomics. The results showed that Th1 and Th2 tropomyosin-induced responses in the spleen are characterized by the unique upregulation of innate (cochlin) and adaptive (Ig κ chain V-III region PC 7175) immune regulators, respectively. In the intestine, tropomyosin allergy concurred with the downregulation of 35 differentiating proteins featuring the overall impairment of metabolic pathways, absorption processes and ammonium ion responses. These data provide new functional biomarkers of tropomyosin-induced immune responses as well as candidate targets for intervention.

SWATH-MS and MRM: Quantification of Ras-related proteins in HIV-1 infected and methamphetamine-exposed human monocyte-derived macrophages (hMDM).

HIV-1 infection of macrophages is a multistep and multifactorial process that has been shown to be enhanced by exposure to methamphetamine (Meth). In this study, we sought to identify the underlying mechanisms of this effect by quantifying the effect of Meth on the proteome of HIV-1-infected macrophages using sequential windowed acquisition of all theoretical fragment ion mass spectra (SWATH-MS) approach. The analyses identified several members of the Rab family of proteins as being dysregulated by Meth treatment, which was confirmed by bioinformatic analyses that indicated substantial alteration of vesicular transport pathways. Validation of the SWATH-MS was performed using an MRM based approach, which confirmed that Meth exposure affects expression of the Rab proteins. However, the pattern of expression changes were highly dynamic, and displayed high donor-to-donor variability. Surprisingly a similar phenomenon was observed for Actin. Our results demonstrate that Meth affects vesicular transport pathways, suggesting a possible molecular mechanism underlying its effect on HIV infection hMDM and a potential broader effect of Meth on cellular homeostasis.

SERUM PROTEOME AND HIGH WATTAGE E-CIGARETTE VAPING: A RANDOMIZED CROSSOVER STUDY

Abstract Objective: Electronic cigarettes (e-cigarettes) are battery-powered devices that aerosolize a liquid (i.e. “e-liquid”) composed of propylene glycol (PG) and/or glycerol (GLY), flavorings and most commonly, nicotine. Suspension of fine particles in a gas forms the aerosol, that is inhaled (i.e. “vaped”). As these small hydrophilic molecules have a short serum/urine half-live, we hypothesized that short-term cessation of vaping in regular users would change serum proteomic signatures. Design and method: Thirty regular e-cigarette users were enrolled in this randomized, investigator-blinded, three-period crossover-study. The periods consisted of nicotine-vaping (nicotine-session), nicotine-free vaping (nicotine-free-session), and complete cessation of vaping (stop-session), all maintained for five days before the session began. Multiparametric metabolomic analyses on urine and serum were used to verify subjects’ protocol compliance, which was 95%. Serum proteomic analysis was performed with Sequential Windowed Acquisition of All theoretical Fragment Ion Mass Spectra (SWATH-MS) Results: Complete cessation of vaping allowed to identify 13 proteins in the serum, which were differentially modulated among sessions. The following proteins of coagulation cascade: anti thrombin III, heparin cofactor II and alpha-2-antiplasmin were higher in the stop-session compared to nicotine- and nicotine-free session. Compared to stop-session, two proteins of complement cascade were lower in the stop-session compared to the nicotine-session. Finally; lower levels of apolipoprotein E were also found in the stop-session compared to the nicotine-free session. (Table 1) Conclusions: In conclusion, our study reveals that short-term e-cigarette cessation in regular users modified several proteins involved in the coagulation pathways, complement cascade and lipid transport. E-cigarette cessation could have a beneficial effect on the cardiovascular health by decreasing the level of proteins involved in the pathogenesis of atherosclerosis, irrespective of the presence of nicotine. This will need further researches in order to confirm our findings in cellular and animal models and to find potential biomarkers of vaping toxicity.

Discrimination of meat from fur-producing and food-providing animals using mass spectrometry-based proteomics

Non-edible meat from fur-producing animals entering into meat consumption chain could pose a serious threat to public health. For the purpose of risk prevention and control of meat safety, in this study, marker peptides for discriminating non-edible meat of fur-producing animals (including fox, silver fox, blue fox, raccoon dog, ussuri raccoon dog, mink and American mink) from meat of food-providing animals (including pig, cattle, sheep and donkey) were explored by shot-gun proteomics and verified by target approach. Two mass spectrometry platforms combined with bioinformatic and chemometric tools were integratedly emloyed for method development. Meat samples were first subjected to in-solution protein digestion and the subsequently tryptic peptides were profiled and quantitated by ultra-high pressure liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q/TOF MS) with sequential windowed acquisition of all theoretical fragment ion mass spectra (SWATH-MS) mode. Candidate marker peptides screened by chemometric tools were further filtered for their biological specificity and detectability through bioinformatics analysis as well as multiple reaction monitoring (MRM) verification with UHPLC-triple quadrupole mass spectrometry (UHPLC-QQQ MS). As a result, 9 peptides, out of 104 candidates, were selected as markers for discriminating analysis, of which DQTLQEELAR was validated as primary indicator of non-edible meat from the concerned fur-producing animals. An MRM method based on the developed marker peptides for routine use was finally proposed for risk alarming of non-edible meat from fur-producing animals in food safety control.

Identification of the differentially expressed proteins in nasopharyngeal carcinoma by proteomics.

BackgroundWe sought to determine the differences with respect to the proteome of nasopharyngeal tissues between patients with nasopharyngeal carcinoma (NPC) and healthy controls by using sequential windowed acquisition of all theoretical fragment ion mass spectra (SWATHTM-MS) and ingenuity pathway analysis (IPA). Our primary purpose was to identify specific protein markers that can be applied for diagnosis or treatment of NPC.MethodsThe CNE-1, CNE-2 and H1299 cell lines were cultured in stable isotope labeling of amino acids in cell culture (SILAC) medium for 10 generations to obtain labeled proteins. Thirty samples of NPC and 30 healthy control nasopharyngeal tissues were collected from the Department of Otolaryngology of the First Affiliated Hospital of Jinan University. Proteome of the nasopharyngeal tissues were analyzed and compared by SWATH-MS to identify differently expressed proteins. Further, extraction of target proteins and biological pathways was performed by IPA. Super-SILAC technique and liquid chromatography-tandem mass spectrometry were used to verify the reliability of the data obtained using SWATH-MS.ResultsWe identified 1,415 differentially expressed proteins between NPC patients and healthy controls. On IPA analysis, EIF2AK2 and MAPK1 proteins were found to be enriched in multiple biological pathways and functional networks.ConclusionsThe differentially expressed proteins EIF2AK2 and MAPK seem to play an important role in the biological network of NPC or may help discover the specific functional proteins of NPC. Further studies are required to identify the pathways and molecular mechanisms that underlie NPC.

Proteomics Approaches for Biomarker and Drug Target Discovery in ALS and FTD.

Neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are increasing in prevalence but lack targeted therapeutics. Although the pathological mechanisms behind these diseases remain unclear, both ALS and FTD are characterized pathologically by aberrant protein aggregation and inclusion formation within neurons, which correlates with neurodegeneration. Notably, aggregation of several key proteins, including TAR DNA binding protein of 43 kDa (TDP-43), superoxide dismutase 1 (SOD1), and tau, have been implicated in these diseases. Proteomics methods are being increasingly applied to better understand disease-related mechanisms and to identify biomarkers of disease, using model systems as well as human samples. Proteomics-based approaches offer unbiased, high-throughput, and quantitative results with numerous applications for investigating proteins of interest. Here, we review recent advances in the understanding of ALS and FTD pathophysiology obtained using proteomics approaches, and we assess technical and experimental limitations. We compare findings from various mass spectrometry (MS) approaches including quantitative proteomics methods such as stable isotope labeling by amino acids in cell culture (SILAC) and tandem mass tagging (TMT) to approaches such as label-free quantitation (LFQ) and sequential windowed acquisition of all theoretical fragment ion mass spectra (SWATH-MS) in studies of ALS and FTD. Similarly, we describe disease-related protein-protein interaction (PPI) studies using approaches including immunoprecipitation mass spectrometry (IP-MS) and proximity-dependent biotin identification (BioID) and discuss future application of new techniques including proximity-dependent ascorbic acid peroxidase labeling (APEX), and biotinylation by antibody recognition (BAR). Furthermore, we explore the use of MS to detect post-translational modifications (PTMs), such as ubiquitination and phosphorylation, of disease-relevant proteins in ALS and FTD. We also discuss upstream technologies that enable enrichment of proteins of interest, highlighting the contributions of new techniques to isolate disease-relevant protein inclusions including flow cytometric analysis of inclusions and trafficking (FloIT). These recently developed approaches, as well as related advances yet to be applied to studies of these neurodegenerative diseases, offer numerous opportunities for discovery of potential therapeutic targets and biomarkers for ALS and FTD.

Changes in the intestinal mucosal proteome of turkeys (Meleagris gallopavo) infected with haemorrhagic enteritis virus

Haemorrhagic enteritis (HE) is a viral disease affecting intestinal integrity and barrier function in turkey (Meleagris gallopavo) and resulting in a significant economic loss. Sequential Windowed Acquisition of All Theoretical Fragment Ion Mass Spectra (SWATH-MS) was applied to identify crucial proteins involved in HE infection. A total of 938 proteins were identified and used to generate a reference library for SWATH-MS analysis. In total, 523 proteins were reliably quantified, and 64 proteins were found to be differentially expressed, including 49 up-regulated and 15 down-regulated proteins between healthy and HE-affected intestinal mucosa. Functional analysis suggested that these proteins were involved in the following categories of cellular pathways and metabolisms: 1) energy pathways; 2) intestine lipid and amino acid metabolism; 3) oxidative stress; 4) intestinal immune response. Major findings of this study demonstrated that natural HE infection is related to the changes in abundance of several proteins involved in cell-intrinsic immune defense against viral invasion, systemic inflammation, modulation of excessive inflammation, B and T cell development and function and antigen presentation. mRNA quantitative expression demonstrated that most of the proteins involved in innate immunity that were found to be differentially abundant were produced by intestinal mucosa, suggesting its direct involvement in immune defences against HE infection.

DIAlignR Provides Precise Retention Time Alignment Across Distant Runs in DIA and Targeted Proteomics

Sequential Windowed Acquisition of All Theoretical Fragment Ion Mass Spectra (SWATH-MS) is widely used for proteomics analysis given its high throughput and reproducibility, but ensuring consistent quantification of analytes across large-scale studies of heterogeneous samples such as human plasma remains challenging. Heterogeneity in large-scale studies can be caused by large time intervals between data acquisition, acquisition by different operators or instruments, and intermittent repair or replacement of parts, such as the liquid chromatography column, all of which affect retention time (RT) reproducibility and, successively, performance of SWATH-MS data analysis. Here, we present a novel algorithm for RT alignment of SWATH-MS data based on direct alignment of raw MS2 chromatograms using a hybrid dynamic programming approach. The algorithm does not impose a chronological order of elution and allows for alignment of elution-order-swapped peaks. Furthermore, allowing RT mapping in a certain window around a coarse global fit makes it robust against noise. On a manually validated dataset, this strategy outperformed the current state-of-the-art approaches. In addition, on real-world clinical data, our approach outperformed global alignment methods by mapping 98% of peaks compared with 67% cumulatively. DIAlignR reduced alignment error up to 30-fold for extremely distant runs. The robustness of technical parameters used in this pairwise alignment strategy is also demonstrated. The source code is released under the BSD license at https://github.com/Roestlab/DIAlignR.

Discrimination of dried sea cucumber (Apostichopus japonicus) products from different geographical origins by sequential windowed acquisition of all theoretical fragment ion mass spectra (SWATH-MS)-based proteomic analysis and chemometrics

The article reports on discrimination of dried sea cucumber Apostichopus japonicus (A. japonicus) products from different geographical regions in China (Xiapu, Jiaonan, Weihai, Yantai, Dalian). For this purpose, sequential window acquisition of all theoretical fragment ion mass spectra (SWATH-MS)-based proteomic analysis combined with chemometrics was evaluated. Proteome variations of A. japonicus samples determined by SWATH-MS using ultra-high performance liquid chromatography coupled to quadrupole time of flight mass spectrometer were mined by chemometric tools to clarify the correlation between protein quantitation data and geographical origin. A differentiating OPLS-DA model was constructed with 55 SWATH-MS quantified proteins. A. japonicus samples from five locations were clearly classified by the developed OPLS-DA model and 100% prediction accuracy was achieved in validation testing as well as real-life sample analysis. Seventeen proteins were identified as discriminatory biomarkers allowing geographical provenance. The proposed method might be applied in origin authentication of commercially dried A. japonicus products.

Discovery of the Consistently Well-Performed Analysis Chain for SWATH-MS Based Pharmacoproteomic Quantification.

Sequential windowed acquisition of all theoretical fragment ion mass spectra (SWATH-MS) has emerged as one of the most popular techniques for label-free proteome quantification in current pharmacoproteomic research. It provides more comprehensive detection and more accurate quantitation of proteins comparing with the traditional techniques. The performance of SWATH-MS is highly susceptible to the selection of processing method. Till now, ≥27 methods (transformation, normalization, and missing-value imputation) are sequentially applied to construct numerous analysis chains for SWATH-MS, but it is still not clear which analysis chain gives the optimal quantification performance. Herein, the performances of 560 analysis chains for quantifying pharmacoproteomic data were comprehensively assessed. Firstly, the most complete set of the publicly available SWATH-MS based pharmacoproteomic data were collected by comprehensive literature review. Secondly, substantial variations among the performances of various analysis chains were observed, and the consistently well-performed analysis chains (CWPACs) across various datasets were for the first time generalized. Finally, the log and power transformations sequentially followed by the total ion current normalization were discovered as one of the best performed analysis chains for the quantification of SWATH-MS based pharmacoproteomic data. In sum, the CWPACs identified here provided important guidance to the quantification of proteomic data and could therefore facilitate the cutting-edge research in any pharmacoproteomic studies requiring SWATH-MS technique.

Mechanisms of Persistence of the Ammonia-Oxidizing Bacteria Nitrosomonas to the Biocide Free Nitrous Acid.

Free nitrous acid (FNA) exerts a broad range of antimicrobial effects on bacteria, although susceptibility varies considerably among microorganisms. Among nitrifiers found in activated sludge of wastewater treatment processes (WWTPs), nitrite-oxidizing bacteria (NOB) are more susceptible to FNA compared to ammonia-oxidizing bacteria (AOB). This selective inhibition of NOB over AOB in WWTPs bypasses nitrate production and improves the efficiency and costs of the nitrogen removal process in both the activated sludge and anaerobic ammonium oxidation (Anammox) system. However, the molecular mechanisms governing this atypical tolerance of AOB to FNA have yet to be understood. Herein we investigate the varying effects of the antimicrobial FNA on activated sludge containing AOB and NOB using an integrated metagenomics and label-free quantitative sequential windowed acquisition of all theoretical fragment ion mass spectra (SWATH-MS) metaproteomic approach. The Nitrosomonas genus of AOB, on exposure to FNA, maintains internal homeostasis by upregulating a number of known oxidative stress enzymes, such as pteridine reductase and dihydrolipoyl dehydrogenase. Denitrifying enzymes were upregulated on exposure to FNA, suggesting the detoxification of nitrite to nitric oxide. Interestingly, proteins involved in stress response mechanisms, such as DNA and protein repair enzymes, phage prevention proteins, and iron transport proteins, were upregulated on exposure to FNA. In addition enzymes involved in energy generation were also upregulated on exposure to FNA. The total proteins specifically derived from the NOB genus Nitrobacter was low and, as such, did not allow for the elucidation of the response mechanism to FNA exposure. These findings give us an understanding of the adaptive mechanisms of tolerance within the AOB Nitrosomonas to the biocidal agent FNA.

Altered Protein Expression of Primary Sea Turtle Cells Exposed to Contaminants Indicates the Potential for In Vitro Proteomics as a High Throughput Tool to Support Biomarker Discovery in Threatened Wildlife

The development of biomarkers of chemical exposure and effect in threatened wildlife is challenging because traditional methods for biomarker discovery that involve in vivo testing or destructive sampling cannot be utilised. These challenges have long since been acknowledged and the development of non‐destructive methods for the detection of biomarkers in wildlife has been examined for many decades. However, despite these efforts, progress in this field has been slow and the development or confirmation of non‐destructive biomarkers of exposure in threatened wildlife has been limited. The usefulness of cell lines along with non‐targeted omics has not been well explored in this context despite the potential for these methods to greatly enhance non‐destructive biomarker development. In order to explore the usefulness of global protein expression of primary cells cultured from wildlife, we optimised exposure and extraction methods and aimed to initially examine the effect of time and dose on global protein expression. Primary green sea turtle (Chelonia mydas) skin cells developed in our lab were exposed to two contaminants known to accumulate in sea turtles ‐ a polychlorinated biphenyl (PCB153) and perfluoronanoic acid (PFNA). Three environmentally relevant concentrations were chosen for exposure (1 μg/L, 0.1 μg/L, and 0.01 μg/L) and cells were exposed for both 24 or 48 hours. The global protein expression was then quantified using Sequential Windowed Acquisition of all Theoretical Fragment Ion Mass Spectra (SWATH‐MS) resulting in over 1000 unique protein identifications. Our results show that a large number of proteins are significantly differentially expressed when sea turtle primary cells are exposed to chemical contaminants. Over 700 proteins were differentially expressed by cells under exposure conditions and time and concentration had significant effects on overall differential expression as well as on the expression if individual proteins. Most significantly, a biomarker of PCB exposure that has previously been identified in sea turtles (superoxide dismutase) was expressed by the skin cells exposed to PCB indicating that these methods can potentially reflect biomarkers measured in whole organisms.Overall, the results from this study provide insight into the effects that time, dose and treatment have on global protein expression of green sea turtles cells, as well as preliminary evidence for the usefulness of non‐targeted proteomics for biomarker discovery. This indicates the potential of the methods described here to support higher throughput confirmation of biomarkers of exposure and effect while reducing the need for invasive sampling on threatened wildlife.Support or Funding InformationFunding support from Griffith University School of Environment and the Australian Rivers InstituteThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Multiplex and Label-Free Relative Quantification Approach for Studying Protein Abundance of Drug Metabolizing Enzymes in Human Liver Microsomes Using SWATH-MS.

We describe a sequential windowed acquisition of all theoretical fragment ion mass spectra (SWATH-MS) based method for label-free, simultaneous, relative quantification of drug metabolism enzymes in human liver microsomes (HLM; n = 78). In-solution tryptic digestion was aided by a pressure cycling method, which allowed a 90 min incubation time, a significant reduction over classical protocols (12-18 h). Digested peptides were separated on an Acquity UHPLC Peptide BEH C18 column using a 60 min gradient method at a flow rate of 0.100 mL/min. The quadrupole-time-of-flight mass spectrometer (ESI-QTOFMS) was operated in positive electrospray ionization mode, and data were acquired by data-dependent acquisition (DDA) and SWATH-MSALL mode. A pooled HLM sample was used as a quality control to evaluate variability in digestion and quantification among different batches, and inter-batch %CV for various proteins was between 3.1 and 7.8%. Spectral library generated from the DDA data identified 1855 distinct proteins and 25 681 distinct peptides at a 1% global false discovery rate (FDR). SWATH data were queried and analyzed for 10 major cytochrome P450 (CYP) enzymes using Skyline, a targeted data extraction software. Further, correlation analysis was performed between functional activity, protein, and mRNA expression for ten CYP enzymes. Pearson correlation coefficient (r) between protein and activity for CYPs ranged from 0.314 (CYP2C19) to 0.767 (CYP2A6). A strong correlation was found between CYP3A4 and CYP3A5 abundance and activity determined using midazolam and testosterone (r > 0.600, p < 0.001). Protein-to-activity correlation was moderate (r > 0.400-0.600, p < 0.001) for CYP1A2, CYP2A6, CYP2B6, CYP2C9, and CYP2E1 and significant but poor (r < 0.400, p < 0.05) for CYP2C8, CYP2C19, and CYP2D6. The findings suggest the suitability of SWATH-MS based method as a valuable and relatively fast analytical technique for relative quantification of proteins in complex biological samples. We also show that protein abundance is a better surrogate than mRNA to predict the activity of CYP activity.