Evaluation of low-density lipoprotein cholesterol equations by cross-platform assessment of accuracy-based EQA data against SI-traceable reference value.

OBJECTIVES: Low-density lipoprotein cholesterol (LDLC) is the primary cholesterol target for the diagnosis and treatment of cardiovascular disease (CVD). Although beta-quantitation (BQ) is the gold standard to determine LDLC levels accurately, many clinical laboratories apply the Friedewald equation to calculate LDLC. As LDLC is an important risk factor for CVD, we evaluated the accuracy of Friedewald and alternative equations (Martin/Hopkins and Sampson) for LDLC. METHODS: We calculated LDLC based on three equations (Friedewald, Martin/Hopkins and Sampson) using the total cholesterol (TC), triglycerides (TG), and high-density lipoprotein cholesterol (HDLC) in commutable serum samples measured by clinical laboratories participating in the Health Sciences Authority (HSA) external quality assessment (EQA) programme over a 5 years period (number of datasets, n=345). LDLC calculated from the equations were comparatively evaluated against the reference values, determined from BQ-isotope dilution mass spectrometry (IDMS) with traceability to the International System of Units (SI). RESULTS: Among the three equations, Martin/Hopkins equation derived LDLC had the best linearity against direct measured (y=1.141x - 14.403; R2=0.8626) and traceable LDLC (y=1.1692x - 22.137; R2=0.9638). Martin/Hopkins equation (R2=0.9638) had the strongest R2 in association with traceable LDLC compared with the Friedewald (R2=0.9262) and Sampson (R2=0.9447) equation. The discordance with traceable LDLC was the lowest in Martin/Hopkins (median=-0.725%, IQR=6.914%) as compared to Friedewald (median=-4.094%, IQR=10.305%) and Sampson equation (median=-1.389%, IQR=9.972%). Martin/Hopkins was found to result in the lowest number of misclassifications, whereas Friedewald had the most numbers of misclassification. Samples with high TG, low HDLC and high LDLC had no misclassification by Martin/Hopkins equation, but Friedewald equation resulted in ∼50% misclassification in these samples. CONCLUSIONS: The Martin/Hopkins equation was found to achieve better agreement with the LDLC reference values as compared to Friedewald and Sampson equations, especially in samples with high TG and low HDLC. Martin/Hopkins derived LDLC also enabled a more accurate classification of LDLC levels.

Global analysis of RNA-binding proteins identifies a positive feedback loop between LARP1 and MYC that promotes tumorigenesis.

In addition to genomic alterations, aberrant changes in post-transcriptional regulation can modify gene function and drive cancer development. RNA-binding proteins (RBPs) are a large class of post-transcriptional regulators that have been increasingly implicated in carcinogenesis. By integrating multi-omics data, we identify LARP1 as one of the most upregulated RBPs in colorectal cancer (CRC) and demonstrate its oncogenic properties. We perform LARP1:RNA interactome profiling and unveil a previously unexplored role for LARP1 in targeting the 3'UTR of oncogenes in CRC. Notably, we identify the proto-oncogenic transcription factor MYC as a key LARP1-regulated target. Our data show that LARP1 positively modulates MYC expression by associating with its 3'UTR. In addition, antisense oligonucleotide-mediated blocking of the interaction between LARP1 and the MYC 3'UTR reduces MYC expression and in vitro CRC growth. Furthermore, a systematic analysis of LARP1:protein interactions reveals IGF2BP3 and YBX1 as LARP1-interacting proteins that also regulate MYC expression and CRC development. Finally, we demonstrate that MYC reciprocally modulates LARP1 expression by targeting its enhancer. In summary, our data reveal a critical, previously uncharacterized role of LARP1 in promoting CRC tumorigenesis, validate its direct regulation of the proto-oncogene MYC and delineate a model of the positive feedback loop between MYC and LARP1 that promotes CRC growth and development.

A comprehensive CHO SWATH-MS spectral library for robust quantitative profiling of 10,000 proteins.

Sequential window acquisition of all theoretical fragment-ion spectra (SWATH) is a data-independent acquisition (DIA) strategy that requires a specific spectral library to generate unbiased and consistent quantitative data matrices of all peptides. SWATH-MS is a promising approach for in-depth proteomic profiling of Chinese hamster Ovary (CHO) cell lines, improving mechanistic understanding of process optimization, and real-time monitoring of process parameters in biologics R&D and manufacturing. However, no spectral library for CHO cells is publicly available. Here we present a comprehensive CHO global spectral library to measure the abundance of more than 10,000 proteins consisting of 199,102 identified peptides from a CHO-K1 cell proteome. The robustness, accuracy and consistency of the spectral library were validated for high confidence in protein identification and reproducible quantification in different CHO-derived cell lines, instrumental setups and downstream processing samples. The availability of a comprehensive SWATH CHO global spectral library will facilitate detailed characterization of upstream and downstream processes, as well as quality by design (QbD) in biomanufacturing. The data have been deposited to ProteomeXchange (PXD016047).

Next Generation Proteomics for Clinical Biomarker Detection Using SWATH-MS.

The technology of "sequential windowed acquisition of all theoretical fragment ion spectra," known as SWATH-MS, is rapidly gaining popularity as a next generation proteomics technology for comprehensive proteome quantitation. In this chapter, we describe the use of SWATH-MS as a label-free quantitative technique in a proteomics study to identify novel serological biomarker for colorectal cancer. We compared the secreted glycoprotein profiles (glyco-secretomes) enriched from the colon adenocarcinoma cell line HCT-116 and its metastatic derivative, E1, and observed that laminin ß-1 (LAMB1) was oversecreted in E1 cells. This novel oversecretion of LAMB1 was validated in colorectal cancer patient serum samples, and ROC analyses showed that LAMB1 performed better than carcinoembryonic antigen (CEA) as a clinical diagnostic biomarker for colorectal cancer. We focus here on the sample preparation methodology and data processing workflow for SWATH-MS studies.

S-Nitrosylation of Divalent Metal Transporter 1 Enhances Iron Uptake to Mediate Loss of Dopaminergic Neurons and Motoric Deficit.

Elevated iron deposition has been reported in Parkinson's disease (PD). However, the route of iron uptake leading to high deposition in the substantia nigra is unresolved. Here, we show a mechanism in enhanced Fe2+ uptake via S-nitrosylation of divalent metal transporter 1 (DMT1). While DMT1 could be S-nitrosylated by exogenous nitric oxide donors, in human PD brains, endogenously S-nitrosylated DMT1 was detected in postmortem substantia nigra. Patch-clamp electrophysiological recordings and iron uptake assays confirmed increased Mn2+ or Fe2+ uptake through S-nitrosylated DMT1. We identified two major S-nitrosylation sites, C23 and C540, by mass spectrometry, and DMT1 C23A or C540A substitutions abolished nitric oxide (NO)-mediated DMT1 current increase. To evaluate in vivo significance, lipopolysaccharide (LPS) was stereotaxically injected into the substantia nigra of female and male mice to induce inflammation and production of NO. The intranigral LPS injection resulted in corresponding increase in Fe2+ deposition, JNK activation, dopaminergic neuronal loss and deficit in motoric activity, and these were rescued by the NO synthase inhibitor l-NAME or by the DMT1-selective blocker ebselen. Lentiviral knockdown of DMT1 abolished LPS-induced dopaminergic neuron loss.SIGNIFICANCE STATEMENT Neuroinflammation and high cytoplasmic Fe2+ levels have been implicated in the initiation and progression of neurodegenerative diseases. Here, we report the unexpected enhancement of the functional activity of transmembrane divalent metal transporter 1 (DMT1) by S-nitrosylation. We demonstrated that S-nitrosylation increased DMT1-mediated Fe2+ uptake, and two cysteines were identified by mass spectrometry to be the sites for S-nitrosylation and for enhanced iron uptake. One conceptual advance is that while DMT1 activity could be increased by external acidification because the gating of the DMT1 transporter is proton motive, we discovered that DMT1 activity could also be enhanced by S-nitrosylation. Significantly, lipopolysaccharide-induced nitric oxide (NO)-mediated neuronal death in the substantia nigra could be ameliorated by using l-NAME, a NO synthase inhibitor, or by ebselen, a DMT1-selective blocker.

Label-Free Quantitative Phosphoproteomics Reveals Regulation of Vasodilator-Stimulated Phosphoprotein upon Stathmin-1 Silencing in a Pair of Isogenic Colorectal Cancer Cell Lines.

In this communication, we present the phosphoproteome changes in an isogenic pair of colorectal cancer cell lines, viz., the poorly metastatic HCT-116 and the highly metastatic derivative E1, upon stathmin-1 (STMN1) knockdown. The aim was to better understand how the alterations of the phosphoproteins in these cells are involved in cancer metastasis. After the phosphopeptides were enriched using the TiO2 HAMMOC approach, comparative proteomics analysis was carried out using sequential window acquisition of all theoretical mass spectra-MS. Following bioinformatics analysis using MarkerView and OneOmics platforms, we identified a list of regulated phosphoproteins that may play a potential role in signaling, maintenance of cytoskeletal structure, and focal adhesion. Among these phosphoproteins, was the actin cytoskeleton regulator protein, vasodilator-stimulated phosphoprotein (VASP), where its change in phosphorylation status was found to be concomitant with STMN1-associated roles in metastasis. We further showed that silencing of stathmin-1 altered the expression, subcellular localization and phosphorylation status of VASP, which suggested that it might be associated with remodeling of the cell cytoskeleton in colorectal cancer metastasis.

Analysis of colorectal cancer glyco-secretome identifies laminin ß-1 (LAMB1) as a potential serological biomarker for colorectal cancer.

The high mortality rate in colorectal cancer is mostly ascribed to metastasis, but the only clinical biomarker available for disease monitoring and prognosis is the carcinoembryonic antigen (CEA). However, the prognostic utility of CEA remains controversial. In an effort to identify novel biomarkers that could be potentially translated for clinical use, we collected the secretomes from the colon adenocarcinoma cell line HCT-116 and its metastatic derivative, E1, using the hollow fiber culture system, and utilized the multilectin affinity chromatography approach to enrich for the secreted glycoproteins (glyco-secretome). The HCT-116 and E1 glyco-secretomes were compared using the label-free quantitative SWATH-MS technology, and a total of 149 glycoproteins were differentially secreted in E1 cells. Among these glycoproteins, laminin ß-1 (LAMB1), a glycoprotein not previously known to be secreted in colorectal cancer cells, was observed to be oversecreted in E1 cells. In addition, we showed that LAMB1 levels were significantly higher in colorectal cancer patient serum samples as compared to healthy controls when measured using ELISA. ROC analyses indicated that LAMB1 performed better than CEA at discriminating between colorectal cancer patients from controls. Moreover, the diagnostic performance was further improved when LAMB1 was used in combination with CEA.

The prognostic value of the stem-like group in colorectal cancer using a panel of immunohistochemistry markers.

Colorectal cancer (CRC) is the second leading cause of cancer-related deaths in the Western world. It is becoming increasingly clear that CRC is a diverse disease, as exemplified by the identification of subgroups of CRC tumours that are driven by distinct biology. Recently, a number of studies have begun to define panels of diagnostically relevant markers to align patients into individual subgroups in an attempt to give information on prognosis and treatment response. We examined the immunohistochemical expression profile of 18 markers, each representing a putative role in cancer development, in 493 primary colorectal carcinomas using tissue microarrays. Through unsupervised clustering in stage II cancers, we identified two cluster groups that are broadly defined by inflammatory or immune-related factors (CD3, CD8, COX-2 and FOXP3) and stem-like factors (CD44, LGR5, SOX2, OCT4). The expression of the stem-like group markers was associated with a significantly worse prognosis compared to cases with lower expression. In addition, patients classified in the stem-like subgroup displayed a trend towards a benefit from adjuvant treatment. The biologically relevant and poor prognostic stem-like group could also be identified in early stage I cancers, suggesting a potential opportunity for the identification of aggressive tumors at a very early stage of the disease.

Unravelling the proteome of degenerative human mitral valves.

Degenerative mitral valve disease (DMVD), which includes the syndromes of mitral valve prolapse (MVP) and flail leaflet, is a common valvular condition which can be complicated by mitral regurgitation and adverse cardiovascular outcomes. Although several genetic and other studies of MVP in dog models have provided some information regarding the underlying disease mechanisms, the proteins and molecular events mediating human MVP pathogenesis have not been unraveled. In this study, we report the first large-scale proteome profiling of mitral valve tissue resected from patients with MVP. A total of 1134 proteins were identified, some of which were validated using SWATH-MS and western blotting. GO annotation of these proteins confirmed the validity of this proteome database in various cardiovascular processes. Among the list of proteins, we found several structural and extracellular matrix proteins, such as asporin, biglycan, decorin, lumican, mimecan, prolargin, versican, and vinculin, that have putative roles in the pathophysiology of MVP. These proteins could also be involved in the cardiac remodeling associated with mitral regurgitation. All MS data have been deposited in the ProteomeXchange with identifier PXD000774 (http://proteomecentral.proteomexchange.org/dataset/PXD000774).

Unbiased proteomic and transcript analyses reveal that stathmin-1 silencing inhibits colorectal cancer metastasis and sensitizes to 5-fluorouracil treatment.

UNLABELLED: Colorectal cancer metastasis is a major cause of mortality worldwide, which may only be controlled with novel methods limiting tumor dissemination and chemoresistance. High stathmin-1 (STMN1) expression was previously established as a hallmark of colorectal cancer progression and predictor of poor survival; however, the mechanism of action is less clear. This work demonstrates that STMN1 silencing arrests tumor-disseminative cascades by inhibiting multiple metastatic drivers, and repressing oncogenic and mesenchymal transcription. Using a sensitive iTRAQ labeling proteomic approach that quantified differential abundance of 4562 proteins, targeting STMN1 expression was shown to reinstate the default cellular program of metastatic inhibition, and promote cellular adhesion via amplification of hemidesmosomal junctions and intermediate filament tethering. Silencing STMN1 also significantly improved chemoresponse to the classical colorectal cancer therapeutic agent, 5FU, via a novel caspase-6 (CASP6)-dependent mechanism. Interestingly, the prometastatic function of STMN1 was independent of p53 but required phosphorylations at S25 or S38; abrogating phosphorylative events may constitute an alternative route to achieving metastatic inhibition. These findings establish STMN1 as a potential target in antimetastatic therapy, and demonstrate the power of an approach coupling proteomics and transcript analyses in the global assessment of treatment benefits and potential side-effects. IMPLICATIONS: Stathmin-1 is a potential candidate in colorectal cancer therapy that targets simultaneously the twin problems of metastatic spread and chemoresistance.

Novel proteomic biomarker panel for prediction of aggressive metastatic hepatocellular carcinoma relapse in surgically resectable patients.

The natural course of early HCC is unknown, and its progression to intermediate and advanced HCC can be diverse. Some early stage HCC patients enjoy prolonged disease-free survival, whereas others suffer aggressive relapse to stage IV metastatic cancer within a year. Comparative proteomics of HCC tumor tissues was carried out using 2D-DIGE and MALDI-TOF/TOF MS to identify proteins that can distinguish these two groups of stage I HCC patients. Twelve out of 148 differentially regulated protein spots were found to differ by approximately 2-fold for the relapse versus nonrelapse patient tissues. Four proteins, namely, heat shock 70 kDa protein 1, argininosuccinate synthase, isoform 2 of UTP-glucose-1-phosphate uridylyltransferase, and transketolase, were shown to have the potential to differentiate metastatic relapse (MR) from nonrelapse (NR) HCC patients after validation by western blotting and immunohistochemical assays. Subsequent TMA analysis revealed a three marker panel of HSP70, ASS1, and UGP2 to be statistically significant in stratifying the two groups of HCC patients. This combination panel achieved high levels of sensitivity and specificity, which has potential for clinical use in identifying HCC tumors prone to MR. This stratification will allow development of clinical management, including close follow-up and possibly treatment options, in the near future.

Prognostic biomarkers for prediction of recurrence of hepatocellular carcinoma: current status and future prospects.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer death worldwide, with region specific etiologies. Despite improvements made in the diagnosis of HCC, the prognosis of HCC patients remains poor due to the high recurrence rate of HCC. There is an urgent need for development of prognostic biomarkers to predict the risk of recurrence in HCC patients after "curative" treatment. Such stratification may aid in patient management and development of personalized medicine for HCC treatment. Omics based studies facilitate the study of global changes in biomolecules in a disease in a high throughput manner, and hence are well poised to understand the complex changes which led to HCC recurrence. The quantitative nature of data obtained from omics based studies allow for development of prognostic biomarkers based on changes in gene, protein and metabolite expression. In this review, we surveyed the application of transcriptomics, proteomics and metabolomics in the study of HCC recurrence. We summarised the data in the literature from these three fields of studies that claimed to be prognostic for HCC recurrence. We critiqued on the limitations of each area of research and the challenges faced in translating the research results for clinical application in predicting HCC recurrence.

iTRAQ analysis of colorectal cancer cell lines suggests Drebrin (DBN1) is overexpressed during liver metastasis.

Colorectal cancer is currently the third in cancer incidence worldwide and the fourth most common cause of cancer deaths. Mortality in colorectal cancer is often ascribed to liver metastasis. In an effort to elucidate the proteins involved in colorectal cancer liver metastasis, we compared the proteome profiles of the human colon adenocarcinoma cell line HCT-116 with its metastatic derivative E1, using the iTRAQ labelling technology, coupled to 2D-LC and MALDI-TOF/TOF MS. A total of 547 proteins were identified, of which 31 of them were differentially expressed in the E1 cell line. Among these proteins, the differential expressions of translationally controlled tumour protein 1, A-kinase anchor protein 12 and Drebrin (DBN1) were validated using Western blot. In particular, DBN1, a protein not previously known to be involved in colorectal cancer metastasis, was found to be overexpressed in E1 as compared to HCT-116 cells. The overexpression of DBN1 was further validated using immunohistochemistry on colorectal cancer tissue sections with matched lymph node and liver metastasis tissues. DBN1 is currently believed to be involved in actin cytoskeleton reorganisation and suppresses actin filament cross-linking and bundling. Since actin reorganisation is an important process for tumour cell migration and invasion, DBN1 may have an important role during colorectal cancer metastasis.

Proteomics discovery of biomarkers for mitral regurgitation caused by mitral valve prolapse.

Mitral regurgitation (MR) is a common valvular lesion frequently caused by mitral valve prolapse (MVP). Surgical intervention in MVP patients with significant MR is predicated on symptoms and measures of left ventricular dysfunction. Because these indicators may be subjective or imprecise, serological biomarkers of disease could be a valuable adjunct to standard evaluation. This study aimed to identify such biomarkers by a proteomics approach. Two pooled plasma samples from 24 MVP subjects with MR (MVP/MR) and 24 non-MVP individuals were treated with the combinatorial peptide ligand library (CPLL) beads prior to iTRAQ labeling and ESI-MS/MS. Lower levels of haptoglobin, platelet basic protein (PBP), and complement component C4b were observed in the MVP/MR as compared to the control sample. These findings were verified by ELISA testing of each of the 24 paired samples, and another 42 matched cases and controls. The AUC values, sensitivities and specificities for (i) haptoglobin, (ii) PBP, (iii) C4b, and (iv) all 3 proteins in combination were (i) 0.813, 76%, 74%; (ii) 0.721, 56%, 77%; (iii) 0.689, 83%, 49%; and (iv) 0.840, 89%, 67%, respectively. In conclusion, haptoglobin, PBP, and C4b are down-regulated in MVP/MR. Their value as serological biomarkers of valvular pathology should be further explored. BIOLOGICAL SIGNIFICANCE: We report the first study that performed comparative proteomics of clinical human plasma samples to identify novel diagnostic biomarkers for mitral valve prolapse (MVP) patients with moderate to severe mitral regurgitation (MR). MR is a common valvular lesion that can be complicated by heart failure, sudden death and atrial fibrillation, yet many patients with severe MR are asymptomatic. Our results revealed reduced levels of haptoglobin, platelet basic protein (PBP), and complement component C4b in the MVP/MR patients as compared to the matched control cases. The plasma proteomics findings were subsequently confirmed by ELISA. Each of these candidate biomarkers has a putative role in the pathophysiology of MVP/MR, further supporting their roles in detection and possibly surveillance and prognostication of this disease.

Enhancing gold recovery from electronic waste via lixiviant metabolic engineering in Chromobacterium violaceum.

Conventional leaching (extraction) methods for gold recovery from electronic waste involve the use of strong acids and pose considerable threat to the environment. The alternative use of bioleaching microbes for gold recovery is non-pollutive and relies on the secretion of a lixiviant or (bio)chemical such as cyanide for extraction of gold from electronic waste. However, widespread industrial use of bioleaching microbes has been constrained by the limited cyanogenic capabilities of lixiviant-producing microorganisms such as Chromobacterium violaceum. Here we show the construction of a metabolically-engineered strain of Chromobacterium violaceum that produces more (70%) cyanide lixiviant and recovers more than twice as much gold from electronic waste compared to wild-type bacteria. Comparative proteome analyses suggested the possibility of further enhancement in cyanogenesis through subsequent metabolic engineering. Our results demonstrated the utility of lixiviant metabolic engineering in the construction of enhanced bioleaching microbes for the bioleaching of precious metals from electronic waste.

Sieving through the cancer secretome.

Cancer is among the most prevalent and serious health problems worldwide. Therefore, there is an urgent need for novel cancer biomarkers with high sensitivity and specificity for early detection and management of the disease. The cancer secretome, encompassing all the proteins that are secreted by cancer cells, is a promising source of biomarkers as the secreted proteins are most likely to enter the blood circulation. Moreover, since secreted proteins are responsible for signaling and communication with the tumor microenvironment, studying the cancer secretome would further the understanding of cancer biology. Latest developments in proteomics technologies have significantly advanced the study of the cancer secretome. In this review, we will present an overview of the secretome sample preparation process and summarize the data from recent secretome studies of six common cancers with high mortality (breast, colorectal, gastric, liver, lung and prostate cancers). In particular, we will focus on the various platforms that were employed and discuss the clinical applicability of the key findings in these studies. This article is part of a Special Issue entitled: An Updated Secretome.

Identification and functional validation of caldesmon as a potential gastric cancer metastasis-associated protein.

In this study, we aim to identify biomarkers for gastric cancer metastasis using a quantitative proteomics approach. The proteins extracted from a panel of 4 gastric cancer cell lines, two derived from primary cancer (AGS, FU97) and two from lymph node metastasis (AZ521, MKN7), were labeled with iTRAQ (8-plex) reagents and analyzed by 2D-LC-MALDI-TOF/TOF MS. In total, 641 proteins were identified with at least a 95% confidence. Using cutoff values of >1.5 and <0.67, 19 proteins were found to be up-regulated and 34 were down-regulated in the metastatic versus primary gastric cancer cell lines respectively. Several of these dysregulated proteins, including caldesmon, were verified using Western blotting. It was found that caldesmon expression was decreased in the two metastasis-derived cell lines, and this was confirmed by further analysis of 7 gastric cancer cell lines. Furthermore, immunohistochemical staining of 9 pairs of primary gastric cancer and the matched lymph node metastasis tissue also corroborated this observation. Finally, knockdown of caldesmon using siRNA in AGS and FU97 gastric cancer cells resulted in an increase in cell migration and invasion, while the overexpression of caldesmon in AZ521 cells led to a decrease in cell migration and invasion. This study has thus established the potential role of caldesmon in gastric cancer metastasis, and further functional studies are underway to delineate the underlying mechanism of action of this protein.

Identification of potential pathways involved in induction of apoptosis by butyrate and 4-benzoylbutyrate in HT29 colorectal cancer cells.

Butyrate and its analogues have long been investigated as potential chemotherapeutic agents. Our previous structure-activity relationship studies of butyrate analogues revealed that 4-benzoylbutyrate had comparable in vitro effects to butyrate when used to treat HT29 and HCT116 colorectal cancer cell lines. The aim of this study was to identify potential mechanisms associated with the antitumorigenic effects of 4-benzoylbutyrate. In this study, butyrate, 3-hydroxybutyrate and 4-benzoylbutyrate were also investigated for their effects on histone deacetylase (HDAC) activity and histone H4 acetylation in HT29 and HCT116 cells. The biological effects of these analogues on HT29 cells were further investigated using quantitative proteomics to determine the proteins potentially involved in their apoptotic and antiproliferative effects. Because 3-hydroxybutyrate had minimal to no effect on apoptosis, proliferation or HDAC activity, this analogue was used to identify differentially expressed proteins that were potentially specific to the apoptotic effects of butyrate and/or 4-benzoylbutyrate. Butyrate treatment inhibited HDAC activity and induced H4 acetylation. 4-Benzoylbutyrate inhibited HDAC activity but failed to enhance H4 acetylation. Proteomic analysis revealed 20 proteins whose levels were similarly altered by both butyrate and 4-benzoylbutyrate. Proteins that showed common patterns of differential regulation in the presence of either butyrate or 4-benzoylbutyrate included c-Myc transcriptional targets, proteins involved in ER homeostasis, signal transduction pathways and cell energy metabolism. Although an additional 23 proteins were altered by 4-benzoylbutyrate uniquely, further work is required to understand the mechanisms involved in its apoptotic effects.

Cancer proteomics.

Cancer presents high mortality and morbidity globally, largely due to its complex and heterogenous nature, and lack of biomarkers for early diagnosis. A proteomics study of cancer aims to identify and characterize functional proteins that drive the transformation of malignancy, and to discover biomarkers to detect early-stage cancer, predict prognosis, determine therapy efficacy, identify novel drug targets, and ultimately develop personalized medicine. The various sources of human samples such as cell lines, tissues, and plasma/serum are probed by a plethora of proteomics tools to discover novel biomarkers and elucidate mechanisms of tumorigenesis. Innovative proteomics technologies and strategies have been designed for protein identification, quantitation, fractionation, and enrichment to delve deeper into the oncoproteome. In addition, there is the need for high-throughput methods for biomarker validation, and integration of the various platforms of oncoproteome data to fully comprehend cancer biology.

Mining the gastric cancer secretome: identification of GRN as a potential diagnostic marker for early gastric cancer.

Gastric cancer is the second leading cause of cancer deaths worldwide, and currently, there are no clinically relevant biomarkers for gastric cancer diagnosis or prognosis. In this study, we applied a 2D-LC-MS/MS based approach, in combination with iTRAQ labeling, to study the secretomes of the gastric cancer cell lines AGS and MKN7. By performing a comparative analysis between the conditioned media and the whole cell lysates, our workflow allowed us to differentiate the bona fide secreted proteins from the intracellular contaminants within the conditioned media. Ninety proteins were found to have higher abundance in the conditioned media as compared to the whole cell lysates of AGS and MKN7 cells. Using a signal peptide and nonclassical secretion prediction tool and an online exosome database, we demonstrated that up to 92.2% of these 90 proteins can be exported out of the cells by classical or nonclassical secretory pathways. We then performed quantitative comparisons of the secretomes between AGS and MKN7, identifying 43 differentially expressed secreted proteins. Among them, GRN was found to be frequently expressed in gastric tumor tissues, but not in normal gastric epithelia by immunohistochemistry. Sandwich ELISA assay also showed elevation of serum GRN levels in gastric cancer patients, particularly those with early gastric cancer. Receiver operating characteristic (ROC) curves analysis confirmed that serum GRN can provide diagnostic discriminations for gastric cancer patients.