A proteomic approach to immune-mediated epithelial-mesenchymal transition

Increasing evidence suggests epithelial-mesenchymal transition (EMT) plays an important role in renal fibrosis. Initial renal injury enables the infiltration of mononuclear cells into the interstitium, and the resulting generation of inflammatory mediators that favour EMT may have a direct role in the development of renal fibrosis. The aim of this study was to investigate the proteome of renal tubular epithelial cells undergoing EMT in vitro. The human proximal tubular cell line (HK-2), exposed to conditioned medium from activated peripheral blood mononuclear cells (PBMC-CM), undergo phenotypic change, from an epithelial towards a fibroblastic phenotype, as evidenced by decreased E-cadherin and increased fibronectin protein expression. Further proteomic analysis, using 2-DE and Progenesis software, revealed the down-regulation of 4 proteins and up-regulation of 23 proteins. MS analysis allowed the positive identification of 15 differentially expressed proteins, including annexin A2, adipocyte plasma membrane-associated protein, T-complex protein 1, reticulocalbin-1 precursor and moesin among others. Western blotting and quantitative real-time PCR confirmed the increase in annexin A2 at the protein and gene level, respectively. Since annexin A2 and S100A6 exist as complexes in B-1 cells, we investigated the S100A6 gene expression further and show an increased expression in HK-2 cells following exposure to activated PBMC-CM. Therefore, we have identified several potential proteins that could play key roles in immune-mediated EMT.     

新型内质网应激诱导蛋白 TRIM25 在乳腺癌细胞中的作用研究

内质网应激是当细胞受到缺氧或营养剥夺等外界因素刺激时而产生的一种效应,该效应与肿 瘤细胞的存活息息相关。该研究揭示了 TRIM25 作为一种新型内质网应激诱导蛋白在肿瘤细胞中所发挥 的作用,可为发现新的肿瘤靶点提供重要依据。该文以乳腺癌细胞 MCF7 为对象,先筛选构建了稳定 敲低 TRIM25 的 MCF7 细胞系;然后,检测了 TRIM25 敲低对内质网应激、未折叠蛋白反应信号通路 和内质网应激诱导的细胞凋亡的影响,以及 TRIM25 在不同乳腺细胞中的表达;最后,通过生物信息学 分析 TRIM25 表达量与乳腺癌患者预后的相关性。结果显示,内质网应激会诱导 TRIM25 表达水平的大 幅上升。通过敲低 TRIM25 可诱导内质网应激、激活未折叠蛋白反应信号通路从而显著促进乳腺癌细胞 MCF7 的凋亡。研究还发现,乳腺原发上皮细胞转化为乳腺癌细胞过程中伴随有 TRIM25 蛋白水平的上 调,生物信息学分析也显示 TRIM25 在乳腺癌组织中高表达,并提示乳腺癌患者预后不良。     

Proteomic and functional characterization of the outer membrane vesicles from the gastric pathogen Helicobacter pylori

The gastric pathogen Helicobacter pylori causes a spectrum of gastro-duodenal diseases, which may be mediated in part by the outer membrane vesicles (OMVs) constitutively shed by the pathogen. We aimed to determine the proteome of H. pylori OMV to help evaluate the mechanisms whereby these structures confer their known immuno-modulatory and cytotoxic activities to host cells, as such disease-associated activities are also conferred by the bacterium from which the vesicles are derived. We also evaluated the effect of the OMV on gastric/colonic epithelial cells, duodenal explants and neutrophils. A proteomic analysis of the OMV proteins separated by SDS-PAGE from two strains of H. pylori (J99 and NCTC 11637) was undertaken and 162 OMV-associated proteins were identified in J99 and 91 in NCTC 11637 by LC-MS/MS. The vesicles are rich in membrane proteins, porins, adhesins and several molecules known to modulate chemokine secretion, cell proliferation and other host cellular processes. Further, the OMVs are also vehicles for the carriage of the cytotoxin-associated gene A cytotoxin in addition to the previously documented toxin, vacuolating cytotoxin. Taken together, it is evident from the proteome of H. pylori OMV that these structures are equipped with the molecules required to interact with host cells in a manner not dissimilar from the intact pathogen.     

The proteome of the human breast cancer cell line MDA-MB-231: Analysis by LTQ-Orbitrap mass spectrometry

We have used a combination of SDS-PAGE and LTQ-Orbitrap MS to explore the proteome of the highly invasive MDA-MB-231 breast cancer cell line. Based on about 520?000 MS/MS spectra, a total of 3481 proteins were identified and subsequently classified according to their cellular distribution and molecular function. Interestingly, a large proportion of proteins (38%) were from cellular membranes and we were able to characterize numerous proteins involved in cancer initiation and progression such as the tumor suppressor p53 and the epidermal growth factor receptor. Together, this study represents the largest proteome database of breast cancer cells realized to date and demonstrates the value of using Orbitrap MS for deeper proteome analysis.     

Differentially expressed proteins of MCF-7 human breast cancer cells affected by Zilongjin, a complementary Chinese herbal medicine

Purpose : Zilongjin, a complementary Chinese herbal medicine, has been used to alleviate the adverse effects of chemotherapeutic drugs in cancer therapy. However, the mechanisms of anti‐cancer activity of Zilongjin are still largely unkonwn. Experimental design : First, the proteomic approach of combined 2‐DE and ESI‐MS/MS was used to investigate the effect of Zilongjin on the protein expression in MCF‐7 cells. Then, the differential expression of some proteins was confirmed by Western blot, cytoimmunofluoresecnce, and quantitative real‐time RT‐PCR analysis. Results : The identified proteins with differential expression, involved in such events as protein translation, cellular signal transduction, cytoskeleton formation and transportation, include seven downregulating proteins, such as Eukaryotic translation initiation factor 3 subunit I, Eukaryotic translation initiation factor 1A Y‐chromosomal, Ran‐specific GTPase‐activating protein, Ubiquitin‐conjugating enzyme E2 N, Tropomodulin‐3, Macrophage‐capping protein, and Tumor protein D52, as well as two upregulating proteins, HSP β‐1 and keratin18. Moreover, the differential expression of three proteins was confirmed. Conclusions and clinical relevance : (i) These results provide a new insight into the molecular mechanisms of Zilongjin on therapy for breast cancer. (ii) The application of the proteomic approaches will result in the more extended appreciation of Chinese medicine than those known at present.     

Towards a comprehensive proteome of normal and malignant human colon tissue by 2-D-LC-ESI-MS and 2-DE proteomics and identification of S100A12 as potential cancer biomarker

The aim of this study was to characterize the proteome of normal and malignant colonic tissue. We previously studied the colon proteome using 2‐DE and MALDI‐MS and identified 734 proteins (Roeßler, M., Rollinger, W., Palme S., Hagmann, M.‐L., et al.., Clin. Cancer Res. 2005, 11, 6550–6557). Here we report the identification of additional colon proteins from the same set of tissue samples using a complementary nano‐flow 2‐D‐LC‐ESI‐MS. In total, 484 proteins were identified in colon. Of these, 252 had also been identified by the 2‐DE/MALDI‐MS approach, whereas 232 proteins were unique to the 2‐D‐LC‐ESI‐MS analysis. Comparing protein expression in neoplastic and normal colon tissue indicated elevated expression of several proteins in colorectal cancer, among them the well established tumor marker carcinoembryonic antigen, as well as calnexin, 40S ribosomal protein S15a, serpin H1, and S100A12. Overexpression of these proteins was confirmed by immunoblotting. Serum levels of S100A12 were determined by ELISA and were found to be strongly elevated in colorectal cancer patients compared to healthy individuals. We conclude, that 2‐D‐LC‐ESI‐MS is a powerful approach to identify and compare protein profiles of tissue samples, that it is complementary to 2‐DE/MALDI‐MS approaches and has the potential to identify novel biomarkers.     

Identification of distinctive protein expression patterns in colorectal adenoma

Purpose: As a pre‐malignant precursor, adenoma provides an ideal tissue for proteome profiling to investigate early colorectal cancer development and provide possible targets for preventive interventions. The aim of this study was to identify patterns of differential protein expression that distinguish colorectal adenoma from normal tissue. Experimental design: Twenty paired samples of adenoma and normal mucosa were analysed by 2‐DE and MALDI‐TOF/TOF MS to detect proteins with ≥2‐fold differential expression. Results: Four proteins were up‐regulated in adenoma (Annexin A3, S100A11, S100P and eIF5A‐1) and three were down‐regulated (Galectin‐1, S100A9 and FABPL). S100P, galectin‐1, S100A9 and FABPL expression was localised by immunohistochemistry. Conclusions and clinical relevance: Distinctive patterns of in vivo protein expression in colorectal adenoma were identified for the first time. These proteins have important functions in cell differentiation, proliferation and metabolism, and may play a crucial role in early colorectal carcinogenesis. The ability to recognise premalignant lesions may have important applications in cancer prevention.     

Proteomic analysis of the response of the human neutrophil-like cell line NB-4 after exposure to anthrax lethal toxin

We used 2‐D DIGE to analyze the early response of NB‐4 cells, a human promyelotic leukemia cell line, exposed to lethal toxin from Bacillus anthracis at the proteome level. After a 2 h exposure, cells were still viable and 43% of spots (n = 1042) showed a significant change in protein level. We identified 59 spots whose expression had changed significantly, and these reflected cytoskeleton damage, mitochondrial lysis and endoplasmic reticulum stress. Actin filament assembly was disrupted as evidenced by an increase in both actin subunits and phosphorylated cofilin, whilst levels of tropomyosin, tropomodulin and actin related protein 2/3 complex subunit decreased. Lower levels of ATP synthase subunits and mitochondrial inner membrane protein were identified as markers of mitochondrial lysis. Levels of various stress response proteins rose and, uniquely, levels of Ca²⁺ binding proteins such as translationally controlled tumor protein rose and hippocalcin‐like protein 1 decreased. This response may have mitigated effects brought about by mitochondrial lysis and endoplasmic reticulum stress, and delayed or prevented apoptosis in NB‐4 cells. These results resemble findings of similar proteomics studies in murine macrophages, although quantitative differences were observed.     

Identification of circulating endorepellin LG3 fragment: Potential use as a serological biomarker for breast cancer

Comparative proteome analysis was performed on the cultured media of human nontumor and malignant breast cell lines, Hs578Bst and Hs578T, respectively, in search of a serological biomarker(s) for breast cancer. Proteins in the conditioned media were separated by 2‐D PAGE and then visualized by silver‐staining. Eight proteins changed differentially by more than two‐fold were identified by MALDI‐TOF/TOF MS. Among the proteins identified, the terminal laminin‐like globular (LG3) domain of endorepellin, which was recently reported as an antiangiogenesis factor, was decreased in the cancer cell line. We confirmed the bone morphogenic protein‐1 (BMP‐1) mediated cleavage site on the N‐terminus of endorepellin LG3 fragment. This finding suggests that the LG3 fragment is specifically released by a BMP‐1 driven limited proteolytic process. The protein was also detected in plasma by Western blot analysis and selected reaction monitoring (SRM). The plasma level of the endorepellin LG3 fragment was significantly lower in breast cancer patients compared to healthy donors (p = 0.017; n = 12). The LG3 protein concentration in the control plasma was measured at approximately 3.7 pmol/mL compared to 1.8 pmol/mL in plasma from the cancer patients. We suggest that these results support the potential use of the endorepellin LG3 fragment as a new serological biomarker for breast cancer.     

iTRAQ‐Based Quantitative Proteomic Analyses of High Grade Esophageal Squamous Intraepithelial Neoplasia

Esophageal squamous cell carcinoma (ESCC) is one of the most common cancers worldwide and is the fourth most lethal cancer in China. Little is known about the proteome of high grade esophageal squamous intraepithelial neoplasia (HGN), which is a premalignant lesion of ESCC. A quantitative proteomic analysis using an isobaric tag for relative and absolute quantification (iTRAQ) approach is used to characterize the protein expression profiles in HGN. Among the 3156 identified proteins, a total of 236 proteins are discovered to be differentially expressed. Compared with paired normal esophageal epithelial tissues, 138 proteins are upregulated and 98 proteins are downregulated in HGN. Bioinformatics analyses are performed according to gene ontology, clusters of orthologous groups, and kyoto encyclopedia of genes and genomes enrichment analyses. Six differentially expressed proteins are chosen and validated by Western blotting. The results of the study increase our understanding of early tumorigenesis during ESCC, and provide insights into the proteome at the initial stages of the disease that can be used to identify potential biomarkers for early diagnosis and for therapeutic targets.     

An evaluation of multidimensional fingerprinting in the context of clinical proteomics

Multidimensional fingerprinting (MDF) utilizes measurable peptide characteristics to identify proteins. In this study, 3‐D fingerprinting, namely, parent protein molecular weight, peptide mass, and peptide retention time on RPLC, is used to identify 331 differentially expressed proteins between normal and human colon cancer plasma membrane samples. A false discovery rate (FDR) procedure is introduced to evaluate the performance of MDF on the colon cancer dataset. This evaluation establishes a false protein identification rate below 15% for this dataset. Western blot analysis is performed to validate the differential expression of the MDF‐identified protein VDAC1 on the original tissue samples. The limits of MDF are further assessed by a simulation study where key parameters such as database size, query size, and mass accuracy are varied. The results of this simulation study demonstrate that fingerprinting with three dimensions yields low FDR values even for large queries on the complete human proteome without the need for prior peptide sequencing by tandem mass spectrometry. Specifically, when mass accuracy is 10 ppm or lower, full human proteome searches can achieve FDR values of 10% or less.     

An application of the 2-nitrobenzenesulfenyl method to proteomic profiling of human colorectal carcinoma: A novel approach for biomarker discovery

In the development of novel biomarkers, the proteomic approach is advantageous because using it the cancer-associated proteins can be directly identified. We previously developed a 2-nitrobenzenesulfenyl (NBS) method to improve quantitative proteome analysis. Here, we applied this method to proteomic profiling of colorectal carcinoma (CRC) to identify novel proteins with altered expression in CRC. Each pair of tumor and normal tissue specimens from 12 CRC patients was analyzed, and approximately 5000 NBS-labeled paired peaks were quantified. Peaks with altered signal intensities (>1.5-fold) and occurring frequently in the samples (>70%) were selected, and 128 proteins were identified by MS/MS analyses as differentially expressed proteins in CRC tissues. Many proteins were newly revealed to be CRC related; 30 were reported in earlier studies of CRC. Six proteins that were up-regulated in CRC (ZYX, RAN, RCN1, AHCY, LGALS1, and VIM) were further characterized and validated by Western blot and immunohistochemistry. All six were found to be CRC-localized, either in cancer cells or in stroma cells near the cancer cells. These results indicate that the proteins identified in this study are novel candidates for CRC markers, and that the NBS method is useful in proteome mining to discover novel biomarkers.     

Comparative proteomic analysis of differentially expressed proteins in an in vitro cellular carcinogenesis model of oral squamous cell carcinoma

In vitro cellular model is an important tool to be used to investigate the cellular events related to pathophysiological conditions in humans. We have developed an in vitro cellular carcinogenesis model of oral squamous cell carcinoma (OSCC). In this study, we performed comparative proteomic analysis using 2‐DE and LC‐tandem mass chromatography to separate and identify differentially expressed proteins. Forty‐five proteins were identified, including 24 proteins with decreased expression and 19 proteins with increased expression during carcinogenesis from immortalized oral epithelial cells to squamous cancerous cells. The identified known proteins were classified into three ontologies of cellular component, molecular function, and biological process. Further validation of five identified proteins (ANXA1, ANXA2, CTSB, KRT17, and S100A6) in the cellular carcinogenesis model and cancerous tissues from OSCC patients confirmed the comparative proteomic results. Moreover, Annexin A1 and A2 expression levels correlated with the pathological differentiation grade of cancerous tissues. Thus, this work provides a dynamic protein file of differentially expressed proteins in oral squamous carcinoma cells, which could provide clues to study the mechanisms of OSCC carcinogenesis and possibly be developed as potential biomarkers for clinical diagnosis or prognostic monitoring.     

Proteomic analysis of breast cancer tissue reveals upregulation of actin-remodeling proteins and its relevance to cancer invasiveness

There is an emerging interest in protein expression profiling with the aim of identifying novel diagnostic markers and therapeutic targets in breast cancer. We analyzed breast cancer tissues by 2-D DIGE using a narrow range IPG strip (pH?5.5-6.7) after the immunodepletion of serum albumin and Ig. Sixty-three protein spots were detected with more than ±1.8-fold differences (p?<0.05 for three technical replicates) from a set of tissue samples in which three tumor and three nontumor samples were randomly selected from six breast cancer subjects and pooled separately. Of these, 53 proteins were successfully identified by MS. Among the proteins whose levels were increased, we identified three novel WD-repeat-motif-bearing proteins that have been known to be involved in actin remodeling: Arp2/3 complex subunit 2 (p34-Arc), coronin-1A and WD-repeat protein 1 (Wdr1). Significantly increased amounts of p34-Arc and coronin-1A in breast cancer were also shown by Western blot analysis of matched tumor and nontumor tissue samples (N?=?11, p?<0.05), and were consistent with the mRNA levels retrieved from publicly available microarray databases. The siRNA knockdown of p34-Arc attenuated the invasion of SK-BR3 breast cancer cells into Matrigel. In contrast, the overexpression of coronin-1A increased this invasive activity. Taken together, the cellular levels of p34-Arc and coronin-1A were linked to cancer development and migration. The data obtained from the present study provides new insight into the management of breast cancer.     

Proteome of human endometrium: Identification of differentially expressed proteins in proliferative and secretory phase endometrium

Purpose: To exploit the potential of proteomics to identify and study additional yet‐unidentified important proteins present in human endometrium. Experimental design: The proteome of human endometrium would be established using 2‐DE and MALDI and the data analyzed to identify differential protein expression in the proliferative and secretory phase of the menstrual cycle using PDQuest software and MALDI. Results: In the present work, 2‐DE of human endometrium protein led to the resolution of over 200 spots. Subsequent MALDI analysis of 215 spots allowed the identification of 194 proteins. A total of 57 out of the 215 spots were found to be differentially expressed, out of which 49 could be identified using MALDI. These differentially expressed proteins included structural proteins, molecular chaperones, signaling proteins, metabolic proteins, proteins related to immunity, RNA biogenesis, protein biosynthesis and others. The differential expressions of seven representative proteins in secretory and proliferative phase endometrium tissue were confirmed by immunoblot analysis. Conclusion and clinical relevance: This study establishes the 2‐D proteome of human endometrium represented by 194 identified protein spots. The present data provides an important clue towards determining the function of these proteins with respect to endometrium related diseases.     

Proteomic characterization of differentially expressed proteins in breast cancer: Expression of hnRNP H1, RKIP and GRP78 is strongly associated with HER-2/neu status

The human epidermal growth factor receptor type 2 (HER‐2/neu) oncoprotein is overexpressed in about 30% of breast cancers and associates with metastatic phenotypes of breast tumours. Dissecting the HER‐2/neu‐modulated molecules in cancer will be helpful in elucidating the underlying molecular mechanisms of HER‐2/neu‐driven tumourigenesis. We investigated the differential proteome profiles between microdissected HER‐2/neu‐positive and ‐negative tumours and unambiguously identified 21 proteins with diverse biological functions by peptide sequencing and NCBInr database interrogation. Six proteins were up‐regulated whereas 15 were down‐regulated in the HER‐2/neu‐positive tumours. Differential expressions of heterogeneous nuclear ribonucleoprotein H1 (hnRNP H1), 78 kDa glucose‐regulated protein (GRP78/Bip) and Raf‐1 kinase inhibitor protein (RKIP), which have not been previously reported as being linked to HER‐2/neu signalling, were further verified. Immunohistochemical staining on tissue microarray sections demonstrated a positive correlation of hnRNP H1 (p = 0.008) and negative correlations of GRP78 and RKIP (p = 0.018 and 0.013, respectively) with HER‐2/neu. Heregulin α1 enhanced hnRNP H1, but reduced GRP78 and RKIP expression in BT474 cells in a dose‐dependent manner, providing evidence of crosstalk between HER‐2/neu signalling and these modulators. Our studies have identified novel modulators that are likely to be intricately involved in HER‐2/neu‐driven tumour proliferation, invasion and metastasis.     

Proteomics cataloging analysis of human expressed prostatic secretions reveals rich source of biomarker candidates

Expressed prostatic secretions (EPS) contain proteins of prostate origin that may reflect the health status of the prostate and be used as diagnostic markers for prostate diseases including prostatitis, benign prostatic hyperplasia, and prostate cancer. Despite their importance and potential applications, a complete catalog of EPS proteins is not yet available. We, therefore, undertook a comprehensive analysis of the EPS proteome using 2‐D micro‐LC combined with MS/MS. Using stringent filtering criteria, we identified a list of 114 proteins with at least two unique‐peptide hits and an additional 75 proteins with only a single unique‐peptide hit. The proteins identified include kallikrein 2 (KLK2), KLK3 (prostate‐specific antigen), KLK11, and nine cluster of differentiation (CD) molecules including CD10, CD13, CD14, CD26, CD66a, CD66c, CD 143, CD177, and CD224. To our knowledge, this list represents the first comprehensive characterization of the EPS proteome, and it provides a candidate biomarker list for targeted quantitative proteomics analysis using a multiple reaction monitoring (MRM) approach. To help prioritize candidate biomarkers, we constructed a protein–protein interaction network of the EPS proteins using Cytoscape (www.cytoscape.org), and overlaid the expression level changes from the Oncomine database onto the network.     

A novel tumor-related protein, C7orf24, identified by proteome differential display of bladder urothelial carcinoma

Proteome analysis of bladder cancer with narrow-range pH 2-DE has identified a novel protein on chromosome 7 encoded by ORF 24 (C7orf24) as one of the highly expressed proteins in cancer cells. C7orf24 is currently registered in the protein database as a hypothetical protein with unknown function. The homologs of C7orf24 in other animals have also been registered as putative protein genes. Western blot analysis using a mAb against C7orf24 confirmed its higher expression in bladder cancer compared with normal tissue. Several other cancer cell lines were also found to express C7orf24. However, the introduction of C7orf24 into Rat-1 or NIH3T3 cells did not cause malignant transformation. A stable transfectant of NIH3T3 cells with recombinant retrovirus vector was produced for a growth rate assay, and a higher growth rate was observed in C7orf24-expressing cells compared with the controls. Six kinds of small interfering RNAs (siRNAs) were then produced, and C7orf24-siRNA#5 showed a strong knockdown effect on protein expression and significant antiproliferative effects on cancer cell lines were demonstrated by the MTT assay. Therefore, C7orf24 may have an important role in cancer cell proliferation, and may be an appropriate therapeutic target molecule against cancer.     

Protein-induced changes during the maturation process of human dendritic cells: A 2-D DIGE approach

Dendritic cells (DCs) are unique antigen presenting cells, which upon maturation change from a specialized antigen‐capturing cell towards a professional antigen presenting cells. In this study, a 2‐D DIGE analysis of immature and mature DCs was performed, to identify proteins changing in expression upon maturation. The protein expression profile of immature and mature DCs, derived from CD14⁺ peripheral blood monocytes was investigated using two pH ranges (pH 4–7 and 6–9) (n = 4). Ninety one differentially expressed spots (p<0.01) were detected, from which we identified 74 spots (81.32%) corresponding to 41 different proteins. The proteins identified play a role in diverse processes, such as antigen processing/presentation, vesicle transport and cytoskeleton remodeling. In addition, a protein interaction network contained 29 (out of 41) proteins, suggesting that, although they functionally originate from distinct classes, these proteins are acting as a protein‐interactome. In conclusion, the proteins shown here to be altered in expression upon maturation are in line with the morphological and functional changes observed during the maturation process, providing a better understanding of the processes involved. This will open new avenues for investigating treatment regimens for immune‐associated disorders.     

Intrinsic subtype-associated changes in the plasma proteome in breast cancer

Breast cancers are classified into five intrinsic subtypes: Luminal subtype A, Luminal subtype B, HER2+, Basal, and Normal-like. In this study, we compared the plasma proteome of patients with Luminal A, Luminal B, HER2+, and Basal subtype with plasma from healthy individuals. Protein changes were considered significant if q-value (false discovery rate) was less than 5%. The highest number of changes in the plasma proteome was observed in patients with Luminal type B followed by Basal type breast cancers. The plasma proteome of Luminal A and HER2+ breast cancer patients did not differ significantly from healthy individuals. In Basal breast cancer, a significant number of plasma proteins were downregulated compared with healthy individuals. Acute phase-response proteins α-glycoprotein orosomucoid 1 and serum amyloid protein P were specifically upregulated in the plasma of Luminal B breast cancer patients, suggesting prevalence of low-grade inflammation. Proteins involved in immune response and free radical scavenging were downregulated in the plasma of Luminal B patients, which is in agreement with defective immune system observed in cancer patients. These results reveal intrinsic subtype specific changes in the plasma proteome that may influence tumor progression as well as the systemic effects of cancer.