Mass Spectrometry-based Proteomic Analysis Research Articles

Cell-type-resolved proteomic analysis of the human liver.

The human liver functions through a complex interplay between parenchymal and non-parenchymal cells. Mass spectrometry-based proteomic analysis of intact tissue has provided an in-depth view of the human liver proteome. However, the predominance of parenchymal cells(hepatocytes) means that the total tissue proteome mainly reflects hepatocyte expression. Here we therefore set out to analyse the proteomes of the major parenchymal and non-parenchymal cell types in the human liver. We applied quantitative label-free proteomic analysis on the major cell types of the human liver: hepatocytes, liver endothelial cells, Kupffer cells and hepatic stellate cells. We identified 9791 proteins, revealing distinct protein expression profiles across cell types, whose in vivo relevance was shown by the presence of cell-type-specific proteins. Analysis of proteins related to the immune system indicated that mechanisms of immune-mediated liver injury include the involvement of several cell types. Furthermore, in-depth investigation of proteins related to the absorption, distribution, metabolism, excretion and toxicity (ADMET) of xenobiotics showed that ADMET-related tasks are not exclusively confined to hepatocytes, and that non-parenchymal cells may contribute to drug transport and metabolism. Overall, the data we provide constitute a unique resource for exploring the proteomes of the major types of human liver cells, which will facilitate an improved understanding of the human liver in health and disease.

MPTP Proteins Regulated by Streptozotocin-Induced Diabetes Mellitus Are Effectively Involved in the Processes of Maintaining Myocardial Metabolic Adaptation.

Mitochondrial permeability transition pores (mPTPs) have become an important topic in investigating the initiation and signaling pathways involved in cardioprotection. Experimental streptozotocin-induced diabetes mellitus (D) was shown to provide sufficient protection to the myocardium via compensatory mechanisms enabling mitochondria to produce energy under pathological conditions during the acute phase. The hypothesized involvement of mPTPs in these processes prompted us to use liquid chromatography and mass spectrometry-based proteomic analysis to investigate the effects of the acute-phase D condition on the structural and regulatory components of this multienzyme complex and the changes caused by compensation events. We detected ADT1, ATP5H, ATPA, and ATPB as the most abundant mPTP proteins. The between-group differences in protein abundance of the mPTP complex as a whole were significantly upregulated in the D group when compared with the control (C) group (p = 0.0106), but fold changes in individual protein expression levels were not significantly altered except for ATP5H, ATP5J, and KCRS. However, none of them passed the criterion of a 1.5-fold change in differential expression for biologically meaningful change. Visualization of the (dis-)similarity between the C and D groups and pairwise correlations revealed different patterns of protein interactions under the C and D conditions which may be linked to endogenous protective processes, of which beneficial effects on myocardial function were previously confirmed.

Physiological and Proteomic Dissection of the Responses of Two Contrasting Wheat Genotypes to Nitrogen Deficiency.

Nitrogen deficiency usually occurs along with aluminum toxicity in acidic soil, which is one of the major constraints for wheat production worldwide. In order to compare adaptive processes to N deficiency with different Al-tolerant wheat cultivars, we chose Atlas 66 and Scout 66 to comprehensively analyze the physiological responses to N deficiency, coupled with label-free mass spectrometry-based proteomics analysis. Results showed that both cultivars were comparable in most physiological indexes under N deficient conditions. However, the chlorophyll content in Scout 66 was higher than that of Atlas 66 under N deficiency. Further proteomic analysis identified 5592 and 5496 proteins in the leaves of Atlas 66 and Scout 66, respectively, of which 658 and 734 proteins were shown to significantly change in abundance upon N deficiency, respectively. The majority of the differentially expressed proteins were involved in cellular N compound metabolic process, photosynthesis, etc. Moreover, tetrapyrrole synthesis and sulfate assimilation were particularly enriched in Scout 66. Our findings provide evidence towards a better understanding of genotype-dependent responses under N deficiency which could help us to develop N efficient cultivars to various soil types.

Hepatic Transporter Alterations by Nuclear Receptor Agonist T0901317 in Sandwich-Cultured Human Hepatocytes: Proteomic Analysis and PBPK Modeling to Evaluate Drug-Drug Interaction Risk

In vitro approaches for predicting drug-drug interactions (DDIs) caused by alterations in transporter protein regulation are not well established. However, reports of transporter regulation via nuclear receptor (NR) modulation by drugs are increasing. This study examined alterations in transporter protein levels in sandwich-cultured human hepatocytes (SCHH; n = 3 donors) measured by liquid chromatography-tandem mass spectrometry-based proteomic analysis after treatment with N-[4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)phenyl]-N-(2,2,2-trifluoroethyl)benzenesulfonamide (T0901317), the first described synthetic liver X receptor agonist. T0901317 treatment (10 μM, 48 hours) decreased the levels of organic cation transporter (OCT) 1 (0.22-, 0.43-, and 0.71-fold of control) and organic anion transporter (OAT) 2 (0.38-, 0.38-, and 0.53-fold of control) and increased multidrug resistance protein (MDR) 1 (1.37-, 1.48-, and 1.59-fold of control). The induction of NR downstream gene expression supports the hypothesis that T0901317 off-target effects on farnesoid X receptor and pregnane X receptor activation are responsible for the unexpected changes in OCT1, OAT2, and MDR1. Uptake of the OCT1 substrate metformin in SCHH was decreased by T0901317 treatment. Effects of decreased OCT1 levels on metformin were simulated using a physiologically-based pharmacokinetic (PBPK) model. Simulations showed a clear decrease in metformin hepatic exposure resulting in a decreased pharmacodynamic effect. This DDI would not be predicted by the modest changes in simulated metformin plasma concentrations. Altogether, the current study demonstrated that an approach combining SCHH, proteomic analysis, and PBPK modeling is useful for revealing tissue concentration-based DDIs caused by unexpected regulation of hepatic transporters by NR modulators. SIGNIFICANCE STATEMENT: This study utilized an approach combining sandwich-cultured human hepatocytes, proteomic analysis, and physiologically based pharmacokinetic modeling to evaluate alterations in pharmacokinetics (PK) and pharmacodynamics (PD) caused by transporter regulation by nuclear receptor modulators. The importance of this approach from a mechanistic and clinically relevant perspective is that it can reveal drug-drug interactions (DDIs) caused by unexpected regulation of hepatic transporters and enable prediction of altered PK and PD changes, especially for tissue concentration-based DDIs.

Comparative Proteomics of Root Apex and Root Elongation Zones Provides Insights into Molecular Mechanisms for Drought Stress and Recovery Adjustment in Switchgrass.

Switchgrass plants were grown in a Sandwich tube system to induce gradual drought stress by withholding watering. After 29 days, the leaf photosynthetic rate decreased significantly, compared to the control plants which were watered regularly. The drought-treated plants recovered to the same leaf water content after three days of re-watering. The root tip (1cm basal fragment, designated as RT1 hereafter) and the elongation/maturation zone (the next upper 1 cm tissue, designated as RT2 hereafter) tissues were collected at the 29th day of drought stress treatment, (named SDT for severe drought treated), after one (D1W) and three days (D3W) of re-watering. The tandem mass tags mass spectrometry-based quantitative proteomics analysis was performed to identify the proteomes, and drought-induced differentially accumulated proteins (DAPs). From RT1 tissues, 6156, 7687, and 7699 proteins were quantified, and 296, 535, and 384 DAPs were identified in the SDT, D1W, and D3W samples, respectively. From RT2 tissues, 7382, 7255, and 6883 proteins were quantified, and 393, 587, and 321 proteins DAPs were identified in the SDT, D1W, and D3W samples. Between RT1 and RT2 tissues, very few DAPs overlapped at SDT, but the number of such proteins increased during the recovery phase. A large number of hydrophilic proteins and stress-responsive proteins were induced during SDT and remained at a higher level during the recovery stages. A large number of DAPs in RT1 tissues maintained the same expression pattern throughout drought treatment and the recovery phases. The DAPs in RT1 tissues were classified in cell proliferation, mitotic cell division, and chromatin modification, and those in RT2 were placed in cell wall remodeling and cell expansion processes. This study provided information pertaining to root zone-specific proteome changes during drought and recover phases, which will allow us to select proteins (genes) as better defined targets for developing drought tolerant plants. The mass spectrometry proteomics data are available via ProteomeXchange with identifier PXD017441.

Host proteome linked to HPV E7-mediated specific gene hypermethylation in cancer pathways

BackgroundHuman papillomavirus (HPV) infection causes around 90% of cervical cancer cases, and cervical cancer is a leading cause of female mortality worldwide. HPV-derived oncoprotein E7 participates in cervical carcinogenesis by inducing aberrant host DNA methylation. However, the targeting specificity of E7 methylation of host genes is not fully understood but is important in the down-regulation of crucial proteins of the hallmark cancer pathways. In this study, we aim to link E7-driven aberrations in the host proteome to corresponding gene promoter hypermethylation events in the hope of providing novel therapeutic targets and biomarkers to indicate the progression of cervical cancer.MethodsHEK293 cells were transfected with pcDNA3.1-E7 plasmid and empty vector and subjected to mass spectrometry-based proteomic analysis. Down-regulated proteins (where relative abundance was determined significant by paired T-test) relevant to cancer pathways were selected as gene candidates for mRNA transcript abundance measurement by qPCR and expression compared with that in SiHa cells (HPV type 16 positive). Methylation Specific PCR was used to determine promoter hypermethylation in genes downregulated in both SiHa and transfected HEK293 cell lines. The FunRich and STRING databases were used for identification of potential regulatory transcription factors and the proteins interacting with transcription factor gene candidates, respectively.ResultsApproximately 400 proteins totally were identified in proteomics analysis. The transcripts of six genes involved in the host immune response and cell proliferation (PTMS, C1QBP, BCAP31, CDKN2A, ZMYM6 and HIST1H1D) were down-regulated, corresponding to proteomic results. Methylation assays showed four gene promoters (PTMS, C1QBP, BCAP31 and CDKN2A) were hypermethylated with 61, 55.5, 70 and 78% increased methylation, respectively. Those four genes can be regulated by the GA-binding protein alpha chain, specificity protein 1 and ETS-like protein-1 transcription factors, as identified from FunRich database predictions.ConclusionsHPV E7 altered the HEK293 proteome, particularly with respect to proteins involved in cell proliferation and host immunity. Down-regulation of these proteins appears to be partly mediated via host DNA methylation. E7 possibly complexes with the transcription factors of its targeting genes and DNMT1, allowing methylation of specific target gene promoters.

Mass Spectrometry for Biomarkers Discovery in Esophageal Squamous Cell Carcinoma.

Mass spectrometry-based proteomics analysis could categorize proteins and study their interactions in large scale in human cancers. By this method, many proteins are upregulated or downregulated in esophageal squamous cell carcinoma (ESCC) when compared to nonneoplastic esophageal mucosae. The method can also be used to identify novel, effective biomarkers for early diagnosis or predict prognosis of patients with ESCC. These changes are associated with different clinical and pathological parameters. Different biological matrices such as pathological tissue, body fluids, and cancer cell lines-based proteomics have widely been used. Herein, we described cell line-based label-free shotgun proteomics (in-solution tryptic digestion) to identify the protein biomarkers differently expressed in ESCC.

Subcellular Proteomics in Conifers: Purification of Nuclei and Chloroplast Proteomes.

The complexity of the plant cell proteome, exhibiting thousands of proteins whose abundance varies in several orders of magnitude, makes impossible to cover most of the plant proteins using standard shotgun-based approaches. Despite this general description of plant proteomes, the complexity is not a big issue (current protocols and instrumentation allow for the identification of several thousand proteins per injection), low or medium abundant proteins cannot be detected most of times, being necessary to fraction or perform targeted analyses in order to detect and quantify them. Among fractioning choices, cell fractioning in its different organelles is a good strategy for gaining not only a deeper coverage of the proteome but also the basis for understanding organelle function, protein dynamics, and trafficking within the cell, as nuclear and chloroplast communication. This approach is used routinely in many labs working with model species; however, the available protocols focusing on tree species are scarce. In this chapter, we provide a simple but robust protocol for isolating nuclei and chloroplasts in pine needles that is fully compatible with later mass spectrometry-based proteome analysis.

Serial in-solution digestion protocol for mass spectrometry-based glycomics and proteomics analysis.

Advancement in mass spectrometry has revolutionized the field of proteomics. However, there remains a gap in the analysis of protein post-translational modifications (PTMs), particularly for glycosylation. Glycosylation, the most common form of PTM, is involved in most biological processes; thus, analysis of glycans along with proteins is crucial to answering important biologically relevant questions. Of particular interest is the brain extracellular matrix (ECM), which has been called the "final Frontier" in neuroscience, which consists of highly glycosylated proteins. Among these, proteoglycans (PGs) contain large glycan structures called glycosaminoglycans (GAGs) that form crucial ECM components, including perineuronal nets (PNNs), shown to be altered in neuropsychiatric diseases. Thus, there is a growing need for high-throughput methods that combine GAG (glycomics) and PGs (proteomics) analysis to unravel the complete biological picture. The protocol presented here integrates glycomics and proteomics to analyze multiple classes of biomolecules. We use a filter-aided sample preparation (FASP) type serial in-solution digestion of GAG classes, including hyaluronan (HA), chondroitin sulfate (CS), and heparan sulfate (HS), followed by peptides. The GAGs and peptides are then cleaned and analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). This protocol is an efficient and economical way of processing tissue or cell lysates to isolate various GAG classes and peptides from the same sample. The method is more efficient (single-pot) than available parallel (multi-pot) release methods, and removal of GAGs facilitates the identification of the proteins with higher peptide-coverage than using conventional-proteomics. Overall, we demonstrate a high-throughput & efficient protocol for mass spectrometry-based glycomic and proteomic analysis (data are available via ProteomeXchange with identifier PXD017513).

Genetic predisposition for increased red blood cell distribution width is an early risk factor for cardiovascular and renal comorbidities.

ABSTRACTRed blood cell distribution width (RDW) is a measurement of the variation in size and volume of red blood cells (RBCs). Increased RDW, indicating a high heterogeneity of RBCs, is prominently associated with a variety of illnesses, especially cardiovascular diseases. However, the significance of this association to the onset and progression of cardiovascular and renal diseases is unknown. We hypothesized that a genetic predisposition for increased RDW is an early risk factor for cardiovascular and renal comorbidities. Since there is no known animal model of increased RDW, we examined a CRISPR/Cas9 gene-edited rat model (RfflTD) that presented with features of hematologic abnormalities as well as severe cardiac and renal comorbidities. A mass spectrometry-based quantitative proteomic analysis indicated anemia of these rats, which presented with significant downregulation of hemoglobin and haptoglobin. Decreased hemoglobin and increased RDW were further observed in RfflTD through complete blood count. Next, a systematic temporal assessment detected an early increased RDW in RfflTD, which was prior to the development of other comorbidities. The primary mutation of RfflTD is a 50 bp deletion in a non-coding region, and our study has serendipitously identified this locus as a novel quantitative trait locus (QTL) for RDW. To our knowledge, our study is the first to experimentally pinpoint a QTL for RDW and provides a novel genetic rat model mimicking the clinical association of increased RDW with poor cardio-renal outcome.

Unravelling the proteomic landscape of extracellular vesicles in prostate cancer by density-based fractionation of urine

ABSTRACT Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular communication and promising diagnostic and prognostic biomarkers in cancer. Despite this enormous clinical potential, the plethora of methods to separate EV from biofluids, providing material of highly variable purity, and lacking knowledge regarding methodological repeatability pose a barrier to clinical translation. Urine is considered an ideal proximal fluid for the study of EV in urological cancers due to its direct contact with the urogenital system. We demonstrate that density-based fractionation of urine by bottom-up Optiprep density gradient centrifugation separates EV and soluble proteins with high specificity and repeatability. Mass spectrometry-based proteomic analysis of urinary EV (uEV) in men with benign and malignant prostate disease allowed us to significantly expand the known human uEV proteome with high specificity and identifies a unique biological profile in prostate cancer not uncovered by the analysis of soluble proteins. In addition, profiling the proteome of EV separated from prostate tumour conditioned medium and matched uEV confirms the specificity of the identified uEV proteome for prostate cancer. Finally, a comparative proteomic analysis with uEV from patients with bladder and renal cancer provided additional evidence of the selective enrichment of protein signatures in uEV reflecting their respective cancer tissues of origin. In conclusion, this study identifies hundreds of previously undetected proteins in uEV of prostate cancer patients and provides a powerful toolbox to map uEV content and contaminants ultimately allowing biomarker discovery in urological cancers.

Modification of cysteine residues for mass spectrometry-based proteomic analysis: facts and artifacts

Mass spectrometric proteomic analysis at the sample preparation stage involves the artificial reduction of disulfide bonds in proteins formed between cysteine residues. Such bonds, when preserved in their native state, complicate subsequent enzymatic hydrolysis and interpretation of the research results. To prevent the re-formation of the disulfide bonds, cysteine residues are protected by special groups, most often by alkylation. In this review, we consider the methods used to modify cysteine residues during sample preparation, as well as possible artifacts of this stage. Particularly, adverse reactions of the alkylating agents with other amino acid residues are described. The most common alkylating compound used to protect cysteine residues in mass spectrometric proteomic analysis is iodoacetamide. However, an analysis of the literature in this area indicates that this reagent causes more adverse reactions than other agents used, such as chloroacetamide and acrylamide. The latter can be recommended for wider use. In the review we also discuss the features of the cysteine residue modifications and their influence on the efficiency of the search for post-translational modifications and protein products of single nucleotide substitutions.

Evaluation of the cytotoxic and cellular proteome impacts of food-grade TiO2 (E171) using simulated gastrointestinal digestions and a tri-culture small intestinal epithelial model

Engineered nanomaterials (ENMs) are widely used in the food industry; however, regulations for ENMs in food are still in the early stages of development due to insufficient health data. This study investigated the cytotoxicity and changes to the proteomic profile in an in vitro small intestinal epithelium model after exposure to digested food models containing the ubiquitous engineered particulate food additive, TiO2 (E171) with an average size around 110 nm. TiO2 at 0.75% or 1.5% (w/w) concentrations in either a fasting food model (FFM) or a standardized food model (SFM) based on American diet were digested using an in vitro oral-gastric-small intestinal simulator, and the resulting digestas were applied to a small intestinal epithelium tri-culture cellular model. Effects on cell layer integrity, cytotoxicity, and oxidative stress were assessed. In order to explore the impact on cellular processes beyond basic cytotoxicity, mass spectrometry-based quantitative proteomic analyses of control and exposed tri-culture cells was performed. TiO2 in FFM, but not in SFM, produced significant, dose-dependent cytotoxicity (24%, p < 0.001), and at the higher dose caused significant oxidative stress (1.24-fold, p < 0.01), indicative of a food matrix effect. No significant perturbations of the cellular proteome were observed with TiO2 in either FFM or SFM food models. However, proteins involved in energy metabolism and protein synthesis were up-regulated by digestas from SFM compared to those from FFM, indicative of a food matrix effect on the cellular proteome. Interestingly, the differences in profiles between the two food models were more pronounced in the presence of TiO2. Together, these results indicate that TiO2 in a fasting diet may be slightly cytotoxic, and that ingested TiO2 does not significantly alter the epithelial proteome, whereas the food matrix alone can have a dramatic effect on the proteome.

Acute pancreatitis promotes the generation of two different exosome populations

Exosomes are small extracellular vesicles that act as intercellular messengers. Previous studies revealed that, during acute pancreatitis, circulating exosomes could reach the alveolar compartment and activate macrophages. However, proteomic analysis suggested that the most likely origin of these exosomes could be the liver instead of the pancreas. The present study aimed to characterize the exosomes released by pancreas to pancreatitis-associated ascitic fluid (PAAF) as well as those circulating in plasma in an experimental model of taurocholate-induced acute pancreatitis in rats. We provide evidence that during acute pancreatitis two different populations of exosomes are generated with relevant differences in cell distribution, protein and microRNA content as well as different implications in their physiological effects. During pancreatitis plasma exosomes, but not PAAF exosomes, are enriched in the inflammatory miR-155 and show low levels of miR-21 and miR-122. Mass spectrometry-based proteomic analysis showed that PAAF exosomes contains 10–30 fold higher loading of histones and ribosomal proteins compared to plasma exosomes. Finally, plasma exosomes have higher pro-inflammatory activity on macrophages than PAAF exosomes. These results confirm the generation of two different populations of exosomes during acute pancreatitis. Deep understanding of their specific functions will be necessary to use them as therapeutic targets at different stages of the disease.

Integrated Transcriptomics, Proteomics, and Glycomics Reveals the Association between Up-regulation of Sialylated N-glycans/Integrin and Breast Cancer Brain Metastasis

Breast cancer brain metastasis has been recognized as one of the central issues in breast cancer research. The elucidation of the processes and pathways that mediate this step will provide important clues for a better understanding of breast cancer metastasis. Increasing evidence suggests that aberrant glycosylation patterns greatly contribute to cell invasion and cancer metastasis. Herein, we combined next-generation RNA sequencing with liquid chromatography-tandem mass spectrometry-based proteomic and N-glycomic analysis from five breast cancer cell lines and one brain cancer cell line to investigate the possible mechanisms of breast cancer brain metastasis. The genes/proteins associated with cell movement were highlighted in breast cancer brain metastasis. The integrin signaling pathway and the up-regulation of α-integrin (ITGA2, ITGA3) were associated with the brain metastatic process. 12 glycogenes showed unique expression in 231BR, which could result in an increase of sialylation during brain metastasis. In agreement with the changes of glycogenes, 60 out of 63 N-glycans that were identified exhibited differential expression among cell lines. The correlation between glycogenes and glycans revealed the importance of sialylation and sialylated glycans in breast cancer brain metastasis. Highly sialylated N-glycans, which were up-regulated in brain-seeking cell line 231BR, likely play a role in brain metastasis.

Bilateral ureteral obstruction is rapidly accompanied by ER stress and activation of autophagic degradation of IMCD proteins, including AQP2.

After the release of bilateral ureteral obstruction (BUO), postobstructive diuresis from an impaired urine concentration mechanism is associated with reduced aquaporin 2 (AQP2) abundance in the inner medullary collecting duct (IMCD). However, the underlying molecular mechanism of this AQP2 reduction is incompletely understood. To elucidate the mechanisms responsible for this phenomenon, we studied molecular changes in IMCDs isolated from rats with 4-h BUO or sham operation at the early onset of AQP2 downregulation using mass spectrometry-based proteomic analysis. Two-hundred fifteen proteins had significant changes in abundances, with 65% of them downregulated in the IMCD of 4-h BUO rats compared with sham rats. Bioinformatic analysis revealed that significantly changed proteins were associated with functional Gene Ontology terms, including “cell-cell adhesion,” “cell-cell adherens junction,” “mitochondrial inner membrane,” “endoplasmic reticulum chaperone complex,” and the KEGG pathway of glycolysis/gluconeogenesis. Targeted liquid chromatography-tandem mass spectrometry or immunoblot analysis confirmed the changes in 19 proteins representative of each predominant cluster, including AQP2. Electron microscopy demonstrated disrupted tight junctions, disorganized adherens junctions, swollen mitochondria, enlargement of the endoplasmic reticulum lumen, and numerous autophagosomes/lysosomes in the IMCD of rats with 4-h BUO. AQP2 and seven proteins chosen as representative of the significantly altered clusters had a significant increase in immunofluorescence-based colocalization with autophagosomes/lysosomes. Immunogold electron microscopy confirmed colocalization of AQP2 with the autophagosome marker microtubule-associated protein 1A/1B-light chain 3 and the lysosomal marker cathepsin D in IMCD cells of rats with 4-h BUO. We conclude that enhanced autophagic degradation of AQP2 and other critical proteins, as well as endoplasmic reticulum stress in the IMCD, are initiated shortly after BUO.

Abstract DP-002: COMPARISON OF POTENTIAL OVARIAN CANCER BIOMARKERS BY MASS SPECTROMETRY-BASED PROTEOMIC ANALYSIS OF RESIDUAL PAP TEST FLUID, CERVICAL SWABS, AND TUMOR TISSUE FROM AN OVARIAN CANCER PATIENT

Abstract Early detection is the key to increased survival for women with ovarian cancer, yet a screening tool has yet to be developed that is adequately sensitive and specific enough for use in the general population. In contrast, screening for cervical cancer by Pap tests has been routinely performed for over 50 years. In the liquid-based Pap test, cells are collected from the cervix and placed into an alcohol-based fixative and then examined for abnormal cells. Since ovarian cancer cells have been observed in Pap tests, we reasoned that ovarian cancer peptide biomarkers may also be present. Our central hypothesis is that proteins shed by ovarian cancer cells can be detected during routine Pap tests by mass spectrometry (MS)-based proteomics. In particular, when collected at the time of cervical cancer Pap test screening, the alcohol-based Pap test fixative and cervical swabs are ideal for biomarker discovery since they are derived from a site near the ovarian cancer (i.e. proteins may be secreted or shed from the tumor and flow through the fallopian tube into the uterus and out the cervical opening). Recently, the fimbria of the fallopian tube have been suggested to be the true precursor to ovarian cancer, strengthening our hypothesis that ovarian cancer proteins will be found in the lower genital tract, perhaps even at early stages. To demonstrate the feasibility of using Pap tests as a biospecimen for proteomics, we previously examined the proteins present in residual Pap test fixative samples from women with normal cervical cytology by MS and described 152 proteins in the “Normal Pap Proteome.” The objective of this study was to identify and compare the proteins from three different sources from the same ovarian cancer patient: (i) the residual Pap test fixative, (ii) a Merocel swab of the cervix, and (iii) the primary ovarian cancer tumor tissue. Proteins were concentrated from the cell-free supernatant of the Pap test fixative or eluted from the swab, and then trypsin digested using the filter-aided sample preparation method. A total protein extract from the patient's tumor tissue was digested by standard in-solution trypsin digestion. The samples were run on 2D-liquid chromatography MS/MS, followed by bioinformatics integration. We identified over 5000 proteins total in the three samples. More than 2000 proteins were expressed in all three ovarian cancer samples, including several known ovarian cancer biomarkers such as CA125. By Scaffold analysis of the Gene Ontology nomenclature of the proteins, we classified the proteins by both cellular localization and biological processes. Additional matched samples from patients will be used to build a library of proteins and peptides that are specific to ovarian cancer for use in the development of targeted MS assays. We conclude that quantification of proteins from Pap test fixatives and cervical swabs will prove to be a rich source of biomarkers for ovarian cancer detection. Citation Format: Kristin L.M. Boylan, Anna C. Rogers, Melissa A. Geller, Peter A. Argenta, Timothy J. Griffin, and Amy P.N. Skubitz. COMPARISON OF POTENTIAL OVARIAN CANCER BIOMARKERS BY MASS SPECTROMETRY-BASED PROTEOMIC ANALYSIS OF RESIDUAL PAP TEST FLUID, CERVICAL SWABS, AND TUMOR TISSUE FROM AN OVARIAN CANCER PATIENT [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr DP-002.

Amyloidosis in surgically resected atrial appendages: a study of 345 consecutive cases with clinical implications.

Histomorphologic parameters of atrial appendages removed during the Cox-Maze procedure have been shown to correlate with recurrence of atrial fibrillation. While amyloid deposition has been noted within atrial appendages, the incidence and significance remains incompletely understood. More accurate amyloid typing methodologies and targeted pharmacotherapeutics have recently been developed, prompting pathologists to provide more detailed information about the type of amyloid identified in such samples. This study sought to fully characterize the morphologic characteristics of atrial amyloid as well as its incidence and clinical significance. Tissue archives were queried for atrial appendages removed during the cardiac surgeries (2010-2014). Patient demographics, imaging features, and salient clinical findings were recorded. Pattern and extent of amyloid deposition were recorded. Typing of the amyloid protein, when present, was performed on a subset of cases by laser capture microdissection with mass spectrometry-based proteomic analysis. A total of 383 atrial appendages from 345 consecutive patients were included in the study (mean age, 69 years; range, 26-92 years). Amyloid was present in 46% of patients. A linear relationship was observed between age and presence of atrial amyloidosis. Women were more likely to have atrial amyloidosis. Two distinct morphologies of amyloid were observed: filamentous and nonfilamentous, and correlated perfectly with amyloid type (filamentous = AANF-type amyloid; nonfilamentous = ATTR-type amyloid). Filamentous deposits were observed in 91% of those with amyloid. Amyloid was more likely to be found in the left atrial appendage than the right. Patients with atrial amyloid, irrespective of type, were more likely to have experienced stroke or TIA and more likely to have atrial arrhythmia preoperatively. Postoperatively, those with atrial amyloid are more likely to experience recurrence of arrhythmia than those who did not have atrial amyloid. Understanding the morphologic characteristics of AANF-type amyloid will allow for identification by the light microscopy and obviates the need for expensive ancillary typing techniques. The finding of nonfilamentous amyloid, should still prompt confirmation of amyloid type so that targeted therapy may be employed.

Isotopically Labeled Clickable Glutathione to Quantify Protein S-Glutathionylation.

Protein S-glutathionylation is one of the important cysteine oxidation events that regulate various redox-mediated biological processes. Despite several existing methods, there are few proteomic approaches to identify and quantify specific cysteine residues susceptible to S-glutathionylation. We previously developed a clickable glutathione approach that labels intracellular glutathione with azido-Ala by using a mutant form of glutathione synthetase. In this study, we developed a quantification strategy with clickable glutathione by using isotopically labeled heavy and light derivatives of azido-Ala, which provides the relative quantification of glutathionylated peptides in mass spectrometry-based proteomic analysis. We applied isotopically labeled clickable glutathione to HL-1 cardiomyocytes, quantifying relative levels of 1398 glutathionylated peptides upon addition of hydrogen peroxide. Importantly, we highlight elevated levels of glutathionylation on sarcomere-associated muscle proteins while validating glutathionylation of two structural proteins, α-actinin and desmin. Our report provides a chemical proteomic strategy to quantify specific glutathionylated cysteines.

High throughput proteomic analysis and machine learning algorithm identifies DUOX2 (dual oxidase 2) as a novel biomarker for response prediction of concurrent chemoradiotherapy for locally advanced rectal cancer.

71 Background: Although many efforts to predict treatment response of concurrent chemoradiotherapy (CCRT) for locally advanced rectal cancer (LARC) have been made, no molecular has proved to be a robust biomarker. Methods: We performed mass spectrometry-based quantitative proteomic analysis of pretreatment Formalin-fixed, Paraffin-embedded (FFPE) biopsy samples of 13 patients with LARC, who were treated with CCRT followed by curative surgery. Based on pathologic report of surgical specimens, we divided thirteen patients as two response groups: complete response (CR) and non-complete response (nCR) groups. Results: A total of 3,637 proteins were identified and 498 proteins were confirmed as expressed at significantly different levels (DEPs; differently expressed proteins) between these two groups. The gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) enrichment analyses were also performed: the result showed that up-regulated DEPs enriched in biological processes (BP) were significantly different between two groups; immune response, cell migration &amp; motility, protein transport in CR group; amide/peptide biosynthetic process, translation, posttranscriptional regulation of gene expression and detoxification in nCR group. To identify the best classifier to evaluate predictive power of signatures, we employed for different machine learning algorithms to classify samples between CR and nCR groups. As a result, we identified the predictive relevance of dual oxidase 2 (DUOX2) as the strongest predictive biomarker. Conclusions: This study identified a new biomarker, DUOX2, applicable to discrimination between CR and nCR after NACRT for LARC. To our knowledge, the present study provides the first identification of a clinical biomarker for response prediction based on in-depth proteomics and machine learning algorithms.