Endometrial carcinoma biomarker discovery and verification using differentially tagged clinical samples with multidimensional liquid chromatography and tandem mass spectrometry

The utility of differentially expressed proteins discovered and identified in an earlier study (DeSouza, L., Diehl, G., Rodrigues, M. J., Guo, J., Romaschin, A. D., Colgan, T. J., and Siu, K. W. M. (2005) Search for cancer markers from endometrial tissues using differentially labeled tags iTRAQ and cleavable ICAT with multidimensional liquid chromatography and tandem mass spectrometry. J. Proteome Res. 4, 377-386) to discriminate malignant and benign endometrial tissue samples was verified in a 40-sample iTRAQ (isobaric tags for relative and absolute quantitation) labeling study involving normal proliferative and secretory samples and Types I and II endometrial cancer samples. None of these proteins had the sensitivity and specificity to be used individually to discriminate between normal and cancer samples. However, a panel of pyruvate kinase, chaperonin 10, and alpha1-antitrypsin achieved the best results with a sensitivity, specificity, predictive value, and positive predictive value of 0.95 each in a logistic regression analysis. In addition, three new potential markers were discovered, whereas two other proteins showed promising trends but were not detected in sufficient numbers of samples to permit statistical validation. Differential expressions of some of these candidate biomarkers were independently verified using immunohistochemistry.     

Multiple reaction monitoring of mTRAQ-labeled peptides enables absolute quantification of endogenous levels of a potential cancer marker in cancerous and normal endometrial tissues

While iTRAQ analyses have proved invaluable for the discovery of potential cancer markers, two outstanding issues that remained were its ineffectiveness to consistently detect specific proteins of interest in a complex sample and to determine the absolute abundance of those proteins. These have been addressed by availability of the mTRAQ reagents (Applied Biosystems, Inc., Foster City, CA) a nonisobaric variant of iTRAQ. We have applied this newly emerging technique to quantify one of our potential markers for endometrial cancer, viz. pyruvate kinase M1/M2. The mTRAQ methodolgy relies on multiple reaction monitoring (MRM) to target tryptic peptides from the protein of interest, thus, ensuring maximal opportunity for detection, while the nonisobaric tags enable specific quantification of each version of the labeled peptides through unique MRM transitions conferred by the labels. Known amounts of synthetic peptides tagged with one of the two available mTRAQ labels, when used as quantification standards in a mixture with the oppositely labeled tryptically digested sample, permit determination of the absolute amounts of the corresponding protein in the sample. The ability to label the sample and reference peptides with either one of the two possible combinations is an inherent advantage of this method, as it provides a means for verification of the reported ratios. In this study, we determined that the amount of pyruvate kinase present in the homogenate from a biopsied EmCa tissue sample was 85 nmol/g of total proteins, while the equivalent concentration in the nonmalignant controls was 21-26 nmol/g of total proteins. This approximately 4-fold higher amount of pyruvate kinase in the cancer sample was further confirmed not only by a direct comparison between the cancer sample and one of the nonmalignant controls, but also independently by an enzyme-linked immunosorbant assay (ELISA). Additionally, the 4-fold higher level of pyruvate kinase amount in the cancer homogenate reported in this study is considerably higher than the 2-fold higher ratio reported across 20 cancer samples in the discovery phase with the iTRAQ technique, suggesting that there exists a possibility that the dynamic range of ratios determined by the iTRAQ technique may have been compressed.     

Identification of novel molecular targets for endometrial cancer using a drill-down LC-MS/MS approach with iTRAQ

Background

The number of patients with endometrial carcinoma (EmCa) with advanced stage or high histological grade is increasing and prognosis has not improved for over the last decade. There is an urgent need for the discovery of novel molecular targets for diagnosis, prognosis and treatment of EmCa, which will have the potential to improve the clinical strategy and outcome of this disease.

Methodology and Results

We used a “drill-down” proteomics approach to facilitate the identification of novel molecular targets for diagnosis, prognosis and/or therapeutic intervention for EmCa. Based on peptide ions identified and their retention times in the first LC-MS/MS analysis, an exclusion list was generated for subsequent iterations. A total of 1529 proteins have been identified below the Proteinpilot® 5% error threshold from the seven sets of iTRAQ experiments performed. On average, the second iteration added 78% new peptides to those identified after the first run, while the third iteration added 36% additional peptides. Of the 1529 proteins identified, only 40 satisfied our criteria for significant differential expression in EmCa in comparison to normal proliferative tissues. These proteins included metabolic enzymes (pyruvate kinase M2 and lactate dehydrogenase A); calcium binding proteins (S100A6, calcyphosine and calumenin), and proteins involved in regulating inflammation, proliferation and invasion (annexin A1, interleukin enhancer-binding factor 3, alpha-1-antitrypsin, macrophage capping protein and cathepsin B). Network analyses revealed regulation of these molecular targets by c-myc, Her2/neu and TNF alpha, suggesting intervention with these pathways may be a promising strategy for the development of novel molecular targeted therapies for EmCa.

Conclusions

Our analyses revealed the significance of drill-down proteomics approach in combination with iTRAQ to overcome some of the limitations of current proteomics strategies. This study led to the identification of a number of novel molecular targets having therapeutic potential for targeted molecular therapies for endometrial carcinoma.     

Comparative study of three proteomic quantitative methods, DIGE, cICAT, and iTRAQ, using 2D gel- or LC-MALDI TOF/TOF

A comparative study on the three quantitative methods frequently used in proteomics, 2D DIGE (difference gel electrophoresis), cICAT (cleavable isotope-coded affinity tags) and iTRAQ (isobaric tags for relative and absolute quantification), was carried out. DIGE and cICAT are familiar techniques used in gel- and LC-based quantitative proteomics, respectively. iTRAQ is a new LC-based technique which is gradually gaining in popularity. A systematic comparison among these quantitative methods has not been reported. In this study, we conducted well-designed comparisons using a six-protein mixture, a reconstituted protein mixture (BSA spiked into human plasma devoid of six abundant proteins), and complex HCT-116 cell lysates as the samples. All three techniques yielded quantitative results with reasonable accuracy when the six-protein or the reconstituted protein mixture was used. In DIGE, accurate quantification was sometimes compromised due to comigration or partial comigration of proteins. The iTRAQ method is more susceptible to errors in precursor ion isolation, which could be manifested with increasing sample complexity. The quantification sensitivity of each method was estimated by the number of peptides detected for each protein. In this regard, the global-tagging iTRAQ technique was more sensitive than the cysteine-specific cICAT method, which in turn was as sensitive as, if not more sensitive than, the DIGE technique. Protein profiling on HCT-116 and HCT-116 p53 -/- cell lysates displayed limited overlapping among proteins identified by the three methods, suggesting the complementary nature of these methods.     

Identification of candidate biomarker proteins released by human endometrial and cervical cancer cells using two-dimensional liquid chromatography/tandem mass spectrometry

Candidate biomarker proteins, including chaperonin 10 and pyruvate kinase, previously discovered and identified using mass-tagging reagents with multidimensional liquid chromatography and tandem mass spectrometry (DeSouza, L.; et al. J. Proteome Res. 2005, 4, 377-386) have been identified in serum-free media of cultured endometrial cancer (KLE and HEC-1-A) and cervical cancer (HeLa) cells. These and other cancer-associated proteins were released by the cultured cells within 24 h of growth. A total of 203 proteins from the KLE cells, 86 from HEC-1-A, and 161 from HeLa are reported.     

Differential expression of novel tyrosine kinase substrates during breast cancer development

To identify novel tyrosine kinase substrates that have never been implicated in cancer, we studied the phosphoproteomic changes in the MCF10AT model of breast cancer progression using a combination of phosphotyrosyl affinity enrichment, iTRAQ technology, and LC-MS/MS. Using complementary MALDI- and ESI-based mass spectrometry, 57 unique proteins comprising tyrosine kinases, phosphatases, and other signaling proteins were detected to undergo differential phosphorylation during disease progression. Seven of these proteins (SPAG9, Toll-interacting protein (TOLLIP), WBP2, NSFL1C, SLC4A7, CYFIP1, and RPS2) were validated to be novel tyrosine kinase substrates. SPAG9, TOLLIP, WBP2, and NSFL1C were further proven to be authentic targets of epidermal growth factor signaling and Iressa (gefitinib). A closer examination revealed that the expression of SLC4A7, a bicarbonate transporter, was down-regulated in 64% of the 25 matched normal and tumor clinical samples. The expression of TOLLIP in clinical breast cancers was heterogeneous with 25% showing higher expression in tumor compared with normal tissues and 35% showing the reverse trend. Preliminary studies on SPAG9, on the other hand, did not show differential expression between normal and diseased states. This is the first time SLC4A7 and TOLLIP have been discovered as novel tyrosine kinase substrates that are also associated with human cancer development. Future molecular and functional studies will provide novel insights into the roles of TOLLIP and SLC4A7 in the molecular etiology of breast cancer.     

Proteome analysis of hepatocellular carcinoma cell strains, MHCC97-H and MHCC97-L, with different metastasis potentials

To better understand the mechanism underlying hepatocellular carcinoma (HCC) metastasis and to search for potential markers for HCC prognosis, differential proteome analysis on two HCC cell strains with high and low metastatic potentials, MHCC97-H and MHCC97-L, was conducted using two-dimensional (2-D) gel electrophoresis followed by matrix-assisted laser desorption/time of flight mass spectrometry and liquid chromatography ion trap mass spectrometry. Image analysis of silver-stained 2-D gels revealed that 56 protein spots showed significant differential expression in MHCC97-H and MHCC97-L cells (Student's t-test, P < 0.05) and 4 protein spots were only detected in MHCC97-H cells. Fourteen protein spots were further identified using in-gel tryptic digestion, peptide mass fingerprinting and tandem mass spectrometry. The expressions of pyruvate kinase M2, ubiquitin carboxy-terminal hydrolase L1, laminin receptor 67 kDa, S100 calcium-binding protein A4, thioredoxin and cytokeratin 19 were elevated in MHCC97-H cells. However, manganese superoxide dismutase, calreticulin precursor, cathepsin D, lactate dehydrogenase B, non-metastatic cell protein 1, cofilin 1 and calumenin precursor were down-regulated in MHCC97-H cells. Intriguingly, most of these identified proteins have been reported to be associated with tumor metastasis. The functional implications of alterations in the levels of these proteins are discussed.     

A strategy for high-resolution protein identification in surface-enhanced laser desorption/ionization mass spectrometry: calgranulin A and chaperonin 10 as protein markers for endometrial carcinoma

Surface-enhanced laser desorption/ionization-mass spectrometry (SELDI-MS) has conventionally been practiced on linear time of flight (TOF) which has low mass accuracy and resolution. Here we demonstrate in an examination of both malignant and nonmalignant endometrial tissue homogenates that high mass accuracy and resolution in the MS stage are crucial. Using a commercially available quadrupole/TOF (QqTOF), we were able to resolve two potential cancer markers, subsequently identified off-line as chaperonin 10 and calgranulin A, that differ by 8 Da in mass. Two off-line protein identification protocols were developed: the first was based on size-exclusion chromatography (SEC), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), protein extraction, trypsin digestion, and matrix-assisted laser desorption/ionization-tandem MS (MALDI-MS/MS); the second on SEC and shotgun nano-liquid chromatography (nanoLC)-MS/MS. Analyses on a cohort of 44 endometrial homogenates showed 22 out of 23 nonmalignant samples had nondetectable to very low abundance of chaperonin 10 and calgranulin A; 17 of the 21 malignant samples had detectable to abundant levels of both proteins. Immunohistochemical staining of a tissue microarray of 32 samples showed that approximately half of malignant endometrial tissues exhibited positive staining for calgranulin A in the malignant epithelium, while 9 out of 10 benign tissues exhibited negative epithelial staining. In addition, macrophages/granulocytes in malignant as well as nonmalignant tissues showed positive staining. No immunostaining occurred in stroma or myometrium. Calgranulin A, in combination with chaperonin 10 and other proteins, may eventually constitute a panel of markers to permit diagnosis and screening of endometrial cancer.     

Quantitative proteome analysis of ovarian cancer tissues using a iTRAQ approach

Quantitative proteomics can be used as a screening tool for identification of differentially expressed proteins as potential biomarkers for cancers. Here, we comparatively analyzed the proteome profiles of ovarian cancer tissues and normal ovarian epithelial tissues. Using the high‐throughput proteomic technology of isobaric tags for relative and absolute quantitation (iTRAQ)‐coupled with two‐dimensional‐liquid chromatography‐tandem mass spectrometry, 1,259 unique proteins were identified. Of those, 205 were potentially differentially expressed between ovarian cancer and normal ovarian tissues. Several of the potentially differentially expressed proteins were validated by Western blotting and real‐time quantitative RT‐PCR analyses. Furthermore, up‐regulation of KRT8, PPA1, IDH2, and S100A11 were validated in ovarian tissue microarrays by immunohistochemistry. Silencing of S100A11 expression suppressed the migration and invasion properties of ovarian cancer cells in vitro. Our study represents the successful application of iTRAQ technology to an investigation of ovarian cancer. Many of the potentially differentially expressed proteins identified had not been linked to ovarian cancer before, and provide valuable novel insights into the underlying mechanisms of carcinogenesis in human ovarian cancer. J. Cell. Biochem. 113: 3762–3772, 2012. © 2012 Wiley Periodicals, Inc.     

Search for potential markers for prostate cancer diagnosis, prognosis and treatment in clinical tissue specimens using amine-specific isobaric tagging (iTRAQ) with two-dimensional liquid chromatography and tandem mass spectrometry

This study aimed to identify candidate new diagnosis and prognosis markers and medicinal targets of prostate cancer (PCa), using state of the art proteomics. A total of 20 prostate tissue specimens from 10 patients with benign prostatic hyperplasia (BPH) and 10 with PCa (Tumour Node Metastasis [TNM] stage T1-T3) were analyzed by isobaric stable isotope labeling (iTRAQ) and two-dimensional liquid chromatography-tandem mass spectrometry (2DLC-MS/MS) approaches using a hybrid quadrupole time-of-flight system (QqTOF). The study resulted in the reproducible identification of 825 nonredundant gene products (p < or = 0.05) of which 30 exhibited up-regulation (> or =2-fold) and another 35 exhibited down-regulation (< or =0.5-fold) between the BPH and PCa specimens constituting a major contribution toward their global proteomic assessment. Selected findings were confirmed by immunohistochemical analysis of prostate tissue specimens. The proteins determined support existing knowledge and uncover novel and promising PCa biomarkers. The PCa proteome found can serve as a useful aid for the identification of improved diagnostic and prognostic markers and ultimately novel chemopreventive and therapeutic targets.     

Expression profiling of more than 3500 proteins of MSS-type colorectal cancer by stable isotope labeling and mass spectrometry

An efficient means of identifying protein biomarkers is essential to proper cancer management. A well-characterized proteome resource holds special promise for the discovery of novel biomarkers. However, quantification of the differences between physiological conditions together with deep down profiling has become increasingly challenging in proteomics. Here, we perform expression profiling of the colorectal cancer (CRC) proteome by stable isotope labeling and mass spectrometry. Quantitative analysis included performing mTRAQ and cICAT labeling in a pooled sample of three microsatellite stable (MSS) type CRC tissues and a pooled sample of their matched normal tissues. We identified and quantified a total of 3688 proteins. Among them, 1487 proteins were expressed differentially between normal and cancer tissues by higher than 2-fold; 1009 proteins showed increased expression in cancer tissue, whereas 478 proteins showed decreased expression. Bioinformatic analysis revealed that our data were largely consistent with known CRC relevant signaling pathways, such as the Wnt/β-catenin, caveolar-mediated endocytosis, and RAN signaling pathways. Mitochondrial dysfunction, known as the Waburg hypothesis, was also confirmed. Therefore, our data showing alterations in the proteomic profile of CRC constitutes a useful resource that may provide insights into tumor progression with later goal of identifying biologically and clinically relevant marker proteins. This article is part of a Special Issue entitled: Proteomics: The clinical link.     

Quantitative protein expression analysis of CLL B cells from mutated and unmutated IgV(H) subgroups using acid-cleavable isotope-coded affinity tag reagents

Relative protein expression levels were compared in leukemic B cells from two patients with chronic lymphocytic leukemia (CLL) having either mutated (M-CLL) or unmutated (UM-CLL) immunoglobulin variable heavy chain genes (IgV(H)). Cells were separated into cytosol and membrane protein fractions then labeled with acid-cleavable ICAT reagents (cICAT). Labeled proteins were digested with trypsin then subjected to SCX and affinity chromatography followed by LC-ESI-MS/MS analysis on a linear ion trap mass spectrometer. A total of 9 proteins from the cytosol fraction and 4 from the membrane fraction showed a 3-fold or greater difference between M-CLL and UM-CLL and a subset of these were examined by Western blot where results concurred with cICAT abundance ratios. The abundance of one of the proteins in particular, the mitochondrial membrane protein cytochrome c oxidase subunit COX G was examined in 6 M-CLL and 6 UM-CLL patients using western blot and results showed significantly greater levels (P < 0.001) in M-CLL patients vs UM-CLL patients. These results demonstrate that stable isotope labeling and mass spectrometry can complement 2D gel electrophoresis and gene microarray technologies for identifying putative and perhaps unique prognostic markers in CLL.     

Identification of glycoproteins associated with different histological subtypes of ovarian tumors using quantitative glycoproteomics

Ovarian cancer is the most lethal gynecologic malignancy in adult women. The origin of epithelial ovarian tumors is both morphologically and biologically heterogeneous, and different subtypes of ovarian tumors have different clinical outcomes. In spite of the heterogeneous nature of ovarian carcinoma, the current biomarkers and treatments for this disease are not subtype-specific. To discover the molecular basis of the ovarian tumor subtypes, we analyzed extracellular glycoproteins of seven common subtypes and normal ovary tissues using quantitative glycoproteomic analysis. Glycoproteins for different ovarian tumor subtypes were identified by liquid chromatography-tandem mass spectrometry and quantitated by spectral counting and then verified by iTRAQ labeling and Western blotting. Glycoproteins uniquely expressed in different subtypes of ovarian tumors or commonly expressed in most subtypes were identified. Using Western blots, we verified that mesothelin was overexpressed in serous carcinoma and transitional-cell carcinoma, CEA5 and CEA6 were overexpressed only in mucinous carcinoma, while versican and periostin were overexpressed in most subtypes of ovarian tumors. This study presents the first proteomic characterization of different ovarian tumor subtypes. The identified glycoproteins for histological subtypes of ovarian tumors will facilitate the understanding of the molecular basis, diagnosis of ovarian tumor subtypes, and predictions for treatment responses to therapeutic agents.     

Proteomic expression analysis of human colorectal cancer: Identification of soluble overexpressed proteins

Colorectal cancer is one of the most common malignancies in developed countries. Scarce clinical signs at the early stages of the disease and the lack of fast and sensitive diagnostic techniques based on the detection of tumor specific protein markers contribute greatly to the high mortality rate. The search for such markers is significantly complicated by the high levels of major structural and cytoskeletal proteins in normal and tumor tissues. Extraction with 0.2 M NaCl in the presence of the nonionic detergent NP-40 was performed to enrich the soluble protein fraction. This modification resulted in a considerably increased sensitivity of detection of minor proteins that may enter the circulation during carcinogenesis. The soluble protein profiles of the paired colon adenocarcinoma and normal tissue specimens were compared using 2D gel electrophoresis, which enabled the detection of 10 proteins whose levels in tumors were elevated at least 10-fold as compared to normal tissue. The proteins were identified by MALDI-TOF mass spectrometry, and two new protein markers of colon cancer, TAF9 and CISH, were discovered. Low levels of CISH synthesis in most normal human tissues and tumors other than colorectal cancer make it a prospective candidate diagnostic marker for this type of cancer.     

Multiplexed quantification of 63 proteins in human urine by multiple reaction monitoring-based mass spectrometry for discovery of potential bladder cancer biomarkers

Three common urological diseases are bladder cancer, urinary tract infection, and hematuria. Seventeen bladder cancer biomarkers were previously discovered using iTRAQ - these findings were verified by MRM-MS in this current study. Urine samples from 156 patients with hernia (n=57, control), bladder cancer (n=76), or urinary tract infection/hematuria (n=23) were collected and subjected to multiplexed LC-MRM/MS to determine the concentrations of 63 proteins that are normally considered to be plasma proteins, but which include proteins found in our earlier iTRAQ study. Sixty-five stable isotope-labeled standard proteotypic peptides were used as internal standards for 63 targeted proteins. Twelve proteins showed higher concentrations in the bladder cancer group than in the hernia and the urinary tract infection/hematuria groups, and thus represent potential urinary biomarkers for detection of bladder cancer. Prothrombin had the highest AUC (0.796), with 71.1% sensitivity and 75.0% specificity for differentiating bladder cancer (n=76) from non-cancerous (n=80) patients. The multiplexed MRM-MS data was used to generate a six-peptide marker panel. This six-peptide panel (afamin, adiponectin, complement C4 gamma chain, apolipoprotein A-II precursor, ceruloplasmin, and prothrombin) can discriminate bladder cancer subjects from non-cancerous subjects with an AUC of 0.814, with a 76.3% positive predictive value, and a 77.5% negative predictive value. This article is part of a Special Section entitled: Understanding genome regulation and genetic diversity by mass spectrometry.     

Verification of endometrial tissue biomarkers previously discovered using mass spectrometry-based proteomics by means of immunohistochemistry in a tissue microarray format

Verification of candidate protein biomarkers is a necessary step in moving from the initial discovery to application. Here, we report results of a verification exercise involving six candidate endometrial cancer biomarkers previously discovered using mass-tagging and multidimensional liquid chromatography/tandem mass spectrometry (DeSouza L., et al. J. Proteome Res. 2005, 4, 377-386) on a cohort of 148 patient samples by means of immunohistochemistry on a tissue microarray format. A panel of the three best-performing biomarkers, chaperonin 10, pyruvate kinase M2, and alpha-1-antitrypsin, achieved a sensitivity of 0.85, specificity of 0.93, predictive value of 0.90, and positive predictive value of 0.88 in discriminating malignant from benign endometrium. The ruggedness of this panel of biomarkers was verified in a 2/3-training-set-1/3-test-set cross-validation analysis by randomly splitting the cohort in 10 ways. The roles of chaperonin 10 and pyruvate kinase M2 in tumorigenesis confirm them as credible cancer biomarkers.     

Protein expression profiling of endometrial malignancies reveals a new tumor marker: chaperonin 10

Endometrial carcinoma is a common malignancy in women, being exceeded in incidence only by that of breast, lung, and colorectal cancers. At present, no serum tumor markers are available for the monitoring of endometrial carcinoma patients, and patients with recurrent disease are detected only following the development of symptoms or abnormalities in imaging assessments. Similarly, no screening tools are available for endometrial carcinoma. Protein profiling by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) has proven to be a sensitive and fast method of analysis for small proteins or peptides to yield specific biomarkers. In this study, a variety of normal and malignant endometrial tissue samples were fractionated and analyzed by SELDI-TOF MS (SELDI is a version of MALDI utilizing protein "chips"). A number of proteins displayed differential expression in malignant endometrial tissues. One of the prominent proteins fractionated by weak cation exchange chromatography and displaying enhanced expression in these malignant tissues was identified as chaperonin 10. The increased expression of chaperonin 10 in malignant endometrial tissues was further confirmed by parallel Western blot and immunohistochemistry analyses.     

Identification of the amyloid β-protein precursor and cystatin C as novel epidermal growth factor receptor regulated secretory proteins in nasopharyngeal carcinoma by proteomics

The epidermal growth factor receptor (EGFR) is usually overexpressed in nasopharyngeal carcinoma (NPC) and is associated with pathogenesis of NPC. However, while EGFR-modulated intracellular proteins have been extensively studied, little is known concerning their extracellular counterparts. To identify EGFR-regulated secreted proteins in NPC, we compared the secretome profiles of TGF-α-stimulated and unstimulated NPC cell line CNE-2. CNE-2 cells were cultured in the absence or presence of TGF-α for 24 h, and secreted proteins were obtained from conditioned serum-free media and enriched by ultrafiltration centrifugation. Using 2-DE and subsequent mass spectrometry, we identified 16 differential secreted proteins, among which the amyloid β-protein precursor (APP) was up-regulated and cystatin C was down-regulated after TGF-α stimulation. We further showed that the secretory changes of APP and cystatin C in CNE-2 after TGF-α stimulation could be abrogated by pretreatment of EGFR tyrosine kinase inhibitor PD153035 and PI3 kinase inhibitor Wortmannin, validating that APP and cystatin C are EGFR-regulated secreted proteins in NPC cells. Immunohistochemistry showed that the expression level of EGFR was positively correlated with the expression level of APP and negatively correlated with the expression level of cystatin C in NPC tissues, indicating that EGFR also regulates expression of APP and cystatin C in clinical NPC tissues. Furthermore, functional analysis showed that the growth and migration of CNE-2 cells decreased after neutralization of secretory APP in the medium using the anti-APP antibody. Our data provide substantial evidence that APP and cystatin C are target secreted proteins of EGFR in NPC, and upregulation of secretory APP by EGFR may be involved in the pathogenesis of NPC.     

Mass spectrometric analysis of protein mixtures at low levels using cleavable 13C-isotope-coded affinity tag and multidimensional chromatography

In order to identify and compare the protein content of very low quantity samples of high complexity, a protocol has been established that combines the differential profiling strength of a new cleavable 13C isotope-coded affinity tag (cICAT) reagent with the high sequence coverage provided by multidimensional liquid chromatography and two modes of tandem mass spectrometry. Major objectives during protocol optimization were to minimize sample losses and establish a robust procedure that employs volatile buffer systems that are highly compatible with mass spectrometry. Cleavable ICAT-labeled tryptic peptides were separated from nonlabeled peptides by avidin affinity chromatography. Subsequently, peptide samples were analyzed by nanoflow liquid chromatography electrospray ionization tandem mass spectrometry and liquid chromatography matrix-assisted laser desorption/ionization tandem mass spectrometry. The use of two ionization/instrumental configurations led to complementary peptide identifications that increased the confidence of protein assignments. Examples that illustrate the power of this strategy are taken from two different projects: i) immunoaffinity purified complexes containing the prion protein from the murine brain, and ii) human tracheal epithelium gland secretions. In these studies, a large number of novel proteins were identified using stringent match criteria, in addition to many that had been identified in previous experiments. In the latter case, the ICAT method produced significant new information on changes that occur in protein expression levels in a patient suffering from cystic fibrosis.     

热CO2气腹处理后结肠癌细胞株COLO 205的蛋白组学变化

目的:研究热CO₂气腹处理后结肠癌细胞株COLO 205的蛋白组学变化,为阐明热CO₂气腹对结肠癌细胞的杀伤作用机制提供依据。方法:用热CO₂气腹处理结肠癌细胞株COLO 205,按有无处理分为处理组与对照组。分别抽提两组细胞的总蛋白质,采用同位素标记的相对和绝对定量（isobaric tags for relative absolute quantitation,iTRAQ）技术标记,液相色谱（LC）分离蛋白质,质谱仪（MS）进行蛋白质鉴定及Western b1ot检测。结果:共筛选得到18个差异表达的蛋白,其中8个蛋白表达上调,10个蛋白表达下调。Western blot显示热休克蛋白HSP70在细胞表达明显高于对照组（P<0.01）;蛋白Myosin-9的表达量在处理后显著下降。结论:热CO₂气腹处理后结肠癌细胞株COLO 205的蛋白组学发生差异性变化。