AGR2, a mucinous ovarian cancer marker, promotes cell proliferation and migration

Ovarian cancer is a leading cause of death in women. Early detection of ovarian cancer is essential to decrease mortality. However, the early diagnosis of ovarian cancer is difficult due to a lack of clinical symptoms and suitable molecular diagnostic markers. Thus, identification of meaningful tumor biomarkers with potential clinical application is clearly needed. To search for a biomarker for the early detection of ovarian cancer, we identified human anterior gradient 2 (AGR2) from our systematic analysis of paired normal and ovarian tumor tissue cDNA microarray. We noted a marked overexpression of AGR2 mRNA and protein in early stage mucinous ovarian tumors compared to normal ovarian tissues and serous type ovarian tumors by Western blot analysis and immunohistochemistry. To further elucidate the role of AGR2 in ovarian tumorigenesis, stable 2774 human ovarian cancer cell lines overexpressing AGR2 were established. Forced expression of AGR2 in 2774 cells enhanced the growth and migration of ovarian cancer cells. AGR2 protein was detected in the serum of mucinous ovarian cancer patients by Western blot and ELISA analysis. Thus, AGR2 is a potential biomarker for the diagnosis of mucinous ovarian cancer and an ELISA assay may facilitate the early detection of mucinous ovarian cancer using patient serum.     

Searching for serum tumor markers for colorectal cancer using a 2‐D DIGE approach

Identification of specific protein markers for colorectal cancer (CRC) could provide a basis for its early diagnosis and detection, as well as clues to the molecular mechanisms governing cancer progression. In the present study, 2‐D DIGE coupled with MS was used to screen for biomarker candidates in the serum proteome of ten human CRC samples and ten healthy control samples. After pooling identical amounts of serum proteins (based on total protein concentration), albumin/IgG was depleted under partially denaturing conditions. Subsequently, the serum samples were labeled with three different CyDyes, and separated by 2‐D DIGE. After analysis with the biological variation analysis module of the DeCyder software, only three spots were found to be significantly elevated in all patient groups (with ratios from 1.52 to 9.08), whereas five spots were significantly down‐regulated in patients (with ratios from ?1.23 to ?10.21) (t‐test; p<0.05). Finally, two potential biomarkers, Transaldolase 1 and thyroid receptor interactor, were chosen for validation and analysis by ELISA with the serum of 30 CRC patients and 30 healthy controls. The serum levels of the two proteins correlated well with the 2‐D DIGE results. Thus, 2‐D DIGE approaches show great promise for biomarker discovery in CRC.     

Proteomics comparison of exosomes from serum and plasma between ultracentrifugation and polymer‐based precipitation kit methods

Exosomes are vesicles with sizes ranging from 30 to 150 nm. The analysis and detection of blood exosomes offers an effective route for cancer diagnosis, prognosis assessment, and therapeutic evaluation of diseases. Due to the difference in separation procedure, collection method and the usage of anticoagulants, serum and plasma samples show diversity test results. In order to evaluate the isolation effect of exosomes in serum and plasma samples, two commonly used exosomal isolation methods, ultracentrifugation and polymer‐based precipitation kit, were used, respectively. And the isolation effects were evaluated by comparing the composition and abundant of proteins from isolated exosomes based on MS‐based proteomics analysis. The results showed that the plasma exosomes extracted by ultracentrifugation identified more exosome biomarkers, and the concentrations of these biomarkers were higher than others. And plasma exosomes could be a better sample for blood‐based proteomics research of exosomes. It would be more useful for future targeted biomarker discovery.     

Cover Picture: Electrophoresis 4'2010

Issue no. 4 is a regular issue with Emphasis on “Bioanalysis”. Part I has 12 articles on bioanalysis featuring methodologies for proteomics, proteins, peptides and nucleic acids. Part II has 5 papers on interactive CE, e.g., MEEKC, MEKC and ACE. The remaining 2 articles make up Part III, and are concerned with the regulation of flow in microfluidics and sensitivity enhancement in CEC. Featured articles include: Identification of candidate biomarkers in ovarian cancer serum by depletion of highly abundant proteins and differential in gel electrophoresis. (( 10.1002/elps.200900441.R1 )) An effective SCAR‐PCR method derived from restriction site amplified polymorphism (RSAP) for the identification of female Schistosoma japonicum of zoonotic significance. (( 10.1002/elps.200900615.R1 )) Rapid and sensitive DNA targets detection using enzyme amplified electrochemical detection based on microchip. (( 10.1002/elps.200900538.R1 ))     

Enrichment of low-abundant serum proteins by albumin/immunoglobulin G immunoaffinity depletion under partly denaturing conditions

We present a simple protocol for affinity depletion to remove the two most abundant serum proteins, albumin and immunoglobulin G (IgG). Under native conditions, albumin/IgG were efficiently removed and several proteins were enriched as shown by two-dimensional electrophoresis (2-DE). Besides that, partly denaturing conditions were established by adding 5 or 20% acetonitrile (ACN) in order to disrupt the binding of low-molecular-weight (LMW) proteins to the carrier proteins albumin/IgG. 2-DE results showed that the total number of detected LMW proteins increased under denaturing conditions when compared to native conditions. Interestingly, the presence of 5% ACN in serum revealed better enrichment of LMW proteins when compared to 20% ACN condition. Seven randomly distributed spots in albumin/IgG depleted serum samples under 5% ACN condition were picked from the 2-DE gels and identified by mass spectrometry (MS). The intensity of five LMW protein spots increased under denaturing conditions when compared to native conditions. Three of the seven identified spots (serum amyloid P, vitamin D-binding protein, and transthyretin) belong to a group of relatively low-abundant proteins, which make up only 1% of all serum proteins. The method presented here improves the resolution of the serum proteome by increasing the number of visualized spots on 2-D gels and allowing the detection and MS identification of LMW proteins and proteins of lower abundance.     

Combinatorial peptide ligand library plasma treatment: Advantages for accessing low‐abundance proteins

Depletion of major blood proteins is one of the most promising approaches to accessing low‐abundance biomarkers. This study compared the use of combinatorial peptide ligand library (CPLL) and albumin and immunoglobulins (IgGs) depletion technology for accessing these low‐abundance proteins in plasma using 2‐DE in an acidic restricted pH range (4–7). Compared with native plasma, both techniques enlarge the visibility of other proteins than albumin and IgG, but there were marked differences in their composition. An increase of the number of spots was detected compared with native plasma (157 spots) with 427 and 557 spots, respectively, detected with albumin and IgG depletion, and CPLL treatment. We selected 70 spots to be identified by MALDI‐TOF related to their absence in the 2‐D gels from native or albumin and IgG‐depleted plasma. The 42 spots identified corresponded to 24 different proteins, with more than half of the proteins which did not belong to the major plasma proteins. CPLL treatment allowed the accessibility to proteolytic fragments obtained from major plasma proteins. We found a large superiority of the CPLL approach over the albumin and IgG depletion process. These findings show the utility of depleting major blood proteins to be able to access low‐abundance proteins and the potential of CPLL to select and identify candidate biomarkers in clinical studies.     

Biomarker discovery in breast cancer serum using 2-D differential gel electrophoresis/ MALDI-TOF/TOF and data validation by routine clinical assays

In the present study, we used 2-D differential gel electrophoresis (2-D DIGE) and MS to screen biomarker candidates in serum samples obtained from 39 patients with breast cancer and 35 controls. First, we pooled the serum samples matched with age and menopausal status. Then, we depleted the two most abundant proteins albumin and IgG by immunoaffinity chromatography under partly denaturing conditions in order to enrich low-abundance proteins and proteins with low molecular weight. Concentrated and desalted samples were labeled with three different CyDyes including one internal standard, pooled from all the samples, and separated with 2-D DIGE in triplicate experiments. Biological variations of the protein expression level were analyzed with DeCyder software and evaluated for reproducibility and statistical significance. The profile of differentially expressed protein spots between patients and controls revealed proapolipoprotein A-I, transferrin, and hemoglobin as up-regulated and three spots, apolipoprotein A-I, apolipoprotein C-III, and haptoglobin alpha2 as down-regulated in patients. Finally, routine clinical immunochemical reactions were used to validate selected candidate biomarkers by quantitative determination of specific proteins in all individual serum samples. The serum level of transferrin correlated well with the 2-D-DIGE results. However, the serum levels of apolipoprotein A-I and haptoglobin could not be detected with the clinical routine diagnostic tests. This demonstrated an advantage 2-D DIGE still has over other techniques. 2-D DIGE can distinguish between isoforms of proteins, where the overall immunochemical quantification does fail due to a lack of isoform-special antibodies.     

Effective protein extraction protocol for proteomics studies of Jerusalem artichoke leaves

Protein extraction is a crucial step for proteomics studies. To establish an effective protein extraction protocol suitable for two‐dimensional electrophoresis (2DE) analysis in Jerusalem artichoke (Helianthus tuberosus L.), three different protein extraction methods—trichloroacetic acid/acetone, Mg/NP‐40, and phenol/ammonium acetate—were evaluated using Jerusalem artichoke leaves as source materials. Of the three methods, trichloroacetic acid/acetone yielded the best protein separation pattern and highest number of protein spots in 2DE analysis. Proteins highly abundant in leaves, such as Rubisco, are typically problematic during leaf 2DE analysis, however, and this disadvantage was evident using trichloroacetic acid/acetone. To reduce the influence of abundant proteins on the detection of low‐abundance proteins, we optimized the trichloroacetic acid/acetone method by incorporating a PEG fractionation approach. After optimization, 363 additional (36.2%) protein spots were detected on the 2DE gel. Our results suggest that trichloroacetic acid/acetone method is a better protein extraction technique than Mg/NP‐40 and phenol/ammonium acetate in Jerusalem artichoke leaf 2DE analysis, and that trichloroacetic acid/acetone method combined with PEG fractionation procedure is the most effective approach for leaf 2DE analysis of Jerusalem artichoke.     

HnRNP K and PDI marked response to chemotherapy to human colorectal cancer cells

5‐Fluorouracil has been the chemotherapy agent of first‐choice for colorectal cancer for many years, but since there are no proven predictors of a patient's response to therapy, all patients receive similar treatment. Consequently, identification of biomarkers for therapeutic effect is crucial for the development of novel therapeutic strategies. Two human colorectal cancer cell lines of different metastatic potential (LoVo and SW480) were studied. IC50 of 5‐FU for both cell lines were measured by 3‐(4,5‐dimethy‐lthiazol‐2‐yl)‐2,5‐diphenyltetrazolium assay and validated by cell cycle analysis. Then the cell lines were treated with 5‐FU at IC50 concentration and protein was extracted for 2‐DE. Differential protein spots were examined by MALDI‐TOF/TOF MS. The expression levels of the different proteins were further confirmed by Western blot and immunofluorescence analyses. Eleven proteins were identified. Expression of heterogeneous nuclear ribonucleoprotein K (hnRNP K) in LoVo cells was higher than in SW480 cells, while protein disulfide isomerase (PDI) displayed the opposite trend. After treatment with 5‐FU, the expression of hnRNP K in LoVo decreased more significantly than in SW480, while PDI in SW480 increased more significantly than in LoVo cells. Conclusion: hnRNP K and PDI in the two cell lines have different expression characteristics. The sensitivity to 5‐FU is not consistent in tumor progression. It may assist in development of novel treatment strategies for colorectal cancer metastasis.     

Aberrant proteins in the saliva of patients with oral squamous cell carcinoma

Confirmation of oral squamous cell cancer (OSCC) currently relies on histological analysis, which does not provide clear indication of cancer development from precancerous lesions. In the present study, whole saliva proteins of patients with OSCC (n = 12) and healthy subjects (n = 12) were separated by 2DE to identify potential candidate biomarkers that are much needed to improve detection of the cancer. The OSCC patients’ 2DE saliva protein profiles appeared unique and different from those obtained from the healthy subjects. The patients’ saliva α1‐antitrypsin (AAT) and haptoglobin (HAP) β chains were resolved into polypeptide spots with increased microheterogeneity, although these were not apparent in their sera. Their 2DE protein profiles also showed presence of hemopexin and α‐1B glycoprotein, which were not detected in the profiles of the control saliva. When subjected to densitometry analysis, significant altered levels of AAT, complement C3, transferrin, transthyretin, and β chains of fibrinogen and HAP were detected. The increased levels of saliva AAT, HAP, complement C3, hemopexin, and transthyretin in the OSCC patients were validated by ELISA. The strong association of AAT and HAP with OSCC was further supported by immunohistochemical staining of cancer tissues. The differently expressed saliva proteins may be useful complementary biomarkers for the early detection and/or monitoring of OSCC, although this requires validation in clinically representative populations.     

Proteomic identification of salivary transferrin as a biomarker for early detection of oral cancer

Oral cancer has a low five-year survival rate. Early detection of oral cancer could reduce the mortality and morbidity associated with this disease. Saliva, which can be sampled non-invasively and is less complex than blood, is a good potential source of oral cancer biomarkers. Proteomic analysis of saliva from oral cancer patients and control subjects was performed to identify salivary biomarkers of early stage oral cancer in humans. The protein profile of pooled salivary samples from patients with oral squamous cell carcinoma (OSCC) or OSCC-free control subjects was analyzed using two-dimensional gel electrophoresis (2DE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analyses. Potential biomarkers were verified by Western blotting and ELISA assays. Transferrin levels were elevated in the saliva of OSCC patients as determined using 2DE followed by MALDI-TOF MS and confirmed by MALDI-TOF/TOF MS, Western blotting and ELISA. The increase in salivary transferrin levels in OSCC patients strongly correlated with the size and stage of the tumor. The area under the receiver-operating characteristics curves showed that salivary transferrin-based ELISA was highly specific, sensitive and accurate for the early detection of oral cancer. We have identified salivary transferrin as a biomarker for the detection of early stage oral cancer. This finding provides a promising basis for the development of a non-invasive diagnostic test for early stage oral cancer.     

Using 2-D electrophoresis followed by nano HPLC in nuclear protein analysis of MCF-7 breast cancer cell line by MALDI-TOF/TOF

The biomarker identification is an important tool in early cancer detection. The MCF-7 breast cancer cell line was chosen as a model system. The nuclear proteins were extracted utilizing a commercially manufactured kit and separated on two dimensional (2-D) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). The first dimension was performed on isoelectric focusing strips with pH range 4–7. Afterwards the proteins were tryptic digested and identification was performed by matrix assisted laser desorption technique with time of flight mass analyzer (MALDI-TOF/TOF). For unambiguous identification proteins with too low concentration or spots contains protein mixture the nano high performance liquid chromatography (HPLC) was used. The 2-D gel electrophoresis (2-DGE) seems to be a good tool to separate large amount of proteins using relatively simple procedure and its hyphenation with HPLC can create the perfect analytical solution for proteome identification. About 150 nuclei protein spots were visualized and the most abundant of them were identified.      

Identification of potential lung cancer biomarkers using an in vitro carcinogenesis model

Lung cancer is one of the deadliest and commonly diagnosed neoplasms. Early diagnosis of this disease is critical for improving clinical outcome and prognosis. Because the early stages of lung cancer often produce no symptoms, it is necessary to identify biomarkers for early detection, prognostic evaluation, and recurrence monitoring of the cancer. To identify potential lung cancer biomarkers, we analyzed the differential protein secretion from transformed bronchial epithelial cells (1198 and 1170-I) as compared to immortalized normal bronchial epithelial cells (BEAS-2B) and non-transformed cells (1799) all of which are derived from BEAS-2B and represent multistage bronchial epithelial carcinogenesis. The proteins recovered from the conditioned media of the cells were separated on two-dimensional gels. There was little difference between the secretome of the BEAS-2B and 1799 cells, whereas the patterns between the transformed 1198 and 1170-I cells and non-transformed 1799 cells were significantly different. Using mass spectrometry and database search, we identified 20 proteins including protein gene product 9.5 (PGP9.5), translationally controlled tumor protein (TCTP), tissue inhibitors of metalloproteinases-2 (TIMP-2), and triosephosphate isomerase (TPI), that were either increased or decreased simultaneously in conditioned media of both 1198 and 1170-I cells. Furthermore, levels of PGP9.5, TCTP, TIMP-2, and TPI were significantly increased not only in the conditioned media of both transformed cell lines when compared to those of BEAS-2B and 1799 cells, but also in plasmas and tissues from lung cancer patients when compared to those in normal controls. We suggest the PGP9.5, TCTP, TIMP-2, and TPI as promising candidates for lung cancer serum biomarkers.     

Comparative proteomic analysis of different Toxoplasma gondii genotypes by two‐dimensional fluorescence difference gel electrophoresis combined with mass spectrometry

Toxoplasma gondii is a protozoan parasite infecting almost all warm‐blooded animals and humans. There are three infective stages of T. gondii: the tachyzoites, the bradyzoites, and the oocysts. The tachyzoite is a rapidly multiplying stage and the main pathogenic factor. In North America and Europe, T. gondii is consisted of four major clonal lineages (namely Types I, II, III, and Type 12). In this study, we explored the proteomic profiles of different genotypes (Type I‐RH strain, Type II‐PRU strain, Type II‐TgQHO strain, and ToxoDB 9‐TgC7 strain) of T. gondii tachyzoites by using 2D DIGE combined with MALDI‐TOF MS. Totally, 110 differentially abundant protein spots were selected. Of these, 98 spots corresponding to 56 proteins from T. gondii were successfully identified. These included surface antigen (SAG1), heat shock protein 70 (Hsp 70), disulfide isomerase, coronin, heat shock protein 60 (Hsp 60), pyruvate kinase, receptor for activated C kinase 1, and peroxiredoxin. Gene ontology enrichment analysis revealed that most of the differentially abundant proteins were involved in biological regulation, metabolic process, response to stress, binding, antioxidant activity, and transporter activity. According to the KEGG metabolic pathway maps of T. gondii, some identified proteins were involved in the glycolytic/gluconeogenesis pathway. The present study identified differentially abundant proteins among different genotypes of T. gondii and these findings have implications for the better understanding of the phenotypic differences among the examined T. gondii genotypes, which in turn may contribute to the better control of toxoplasmosis.     

Isotope coded protein label quantification of serum proteins—Comparison with the label-free LC-MS and validation using the MRM approach

Protein quantification based upon mass spectrometry is gaining ground in diverse applications of biological and clinical relevance. The present article focuses on one of the most complex biological fluids - serum - and provides a novel ICPL based quantification protocol. The results are compared to a label-free (data independent alternate scanning) absolute quantification method. The validation is performed using MRM based protein quantification technique. Regarding the ICPL approach, serum samples used in this study were depleted of high abundant proteins, labeled with ICPL and fractionated according to their respective pI (3-5, 5-7 and 7-12). The samples were further subjected to tryptic digestion followed by treatment with the Glu-C enzyme. The peptides were analyzed on a 2D-nano-LC system using four different concentrations of salt injections (45, 75, 150 and 500 mM ammonium acetate). The LC system was connected on-line with the electrospray ion-trap mass spectrometer. For the label-free quantification the serum samples were depleted and digested with trypsin. A proteome-wide comparison was performed using highly reproducible LC and data independent alternate scanning in conjunction with a high mass accuracy orthogonal time-of-flight mass spectrometer. Selected proteins, found by both methods, were validated using the MRM approach. For this purpose non-depleted tryptically digested serum samples were analyzed by LC coupled with a triple-quadrupole MS. The relative protein quantification using ICPL and mass spectrometry allowed for the detection of approximately 200 proteins, whereas about 2/3 of those contained the ICPL label and could therefore be quantified. Label-free approach used no fractionation, less sample and was able to identify and quantify over 110 proteins. The identified proteins covered generally 3-4 orders of magnitude of protein concentration in human serum. Changes in relative abundance of eight proteins were validated using MRM. This study, for the first time, shows the ability of the relative protein quantification based upon ICPL and 2D-LC-MS/MS to quantify serum biomarkers. It provides two additional label-free approaches that could validate and bring additional value to the label-based results, offering a starting point for comprehensive proteomics studies aiming at revealing biomarkers of clinical relevance.     

Mass Spectrometric Characterization of Protein Structure Details Refines the Proteome Signature for Invasive Ductal Breast Carcinoma

Early diagnosis as well as individualized therapies are necessary to reduce the mortality of breast cancer, and personalized patient care strategies rely on novel prognostic or predictive factors. In this study, with six breast cancer patients, 2D gel analysis was applied for studying protein expression differences in order to distinguish invasive ductal breast carcinoma, the most frequent breast tumor subtype, from control samples. In total, 1203 protein spots were assembled in a 2D reference gel. Differentially abundant spots were subjected to peptide mass fingerprinting for protein identification. Twenty proteins with their corresponding 38 differentially expressed 2D gel spots were contained in our previously reported proteome signature, suggesting that distinct protein forms were contributing. In-depth MS/MS measurements enabled analyses of protein structure details of selected proteins. In protein spots that significantly contributed to our signature, we found that glyceraldehyde-3-phosphate dehydrogenase was N-terminally truncated, pyruvate kinase M2 and nucleoside diphosphate kinase A but not other isoforms of these proteins were of importance, and nucleophosmin phosphorylation at serine residues 106 and 125 were clearly identified. Principle component analysis and hierarchical clustering with normalized quantitative data from the 38 spots resulted in accurate separation of tumor from control samples. Thus, separation of tissue samples as in our initial proteome signature could be confirmed even with a different proteome analysis platform. In addition, detailed protein structure investigations enabled refining our proteome signature for invasive ductal breast carcinoma, opening the way to structure-/function studies with respect to disease processes and/or therapeutic intervention.     

An approach to remove alpha amylase for proteomic analysis of low abundance biomarkers in human saliva

Proteomic characterization of human whole saliva for the identification of disease-specific biomarkers is guaranteed to be an easy-to-use and powerful diagnostic tool for defining the onset, progression and prognosis of human systemic diseases and, in particular, oral diseases. The high abundance of proteins, mainly alpha amylase, hampers the detection of low abundant proteins appearing in the disease state and therefore should be removed. In the present study a 2-DE was used to analyze human whole saliva following the removal of alpha amylase by affinity adsorption to potato starch. After alpha amylase removal whole saliva was analyzed by SDS-PAGE showing at least sixfold removal efficiency and by an alpha amylase activity assay showing 97% reduced activity. MS identification of the captured alpha amylase after elution demonstrated specific removal; 2-DE analysis showed the selective removal of alpha amylase and consequently increased gel resolution. MS identification of protein spots in the 60 kDa area revealed 15 proteins, which were masked before alpha amylase removal. In conclusion, treatment of human whole saliva with an alpha amylase removal device increases gel resolution and enables a higher protein sample for analysis.     

An effective skeletal muscle prefractionation method to remove abundant structural proteins for optimized two-dimensional gel electrophoresis

Proteomic analysis of biological samples in disease models or therapeutic intervention studies requires the ability to detect and identify biologically relevant proteins present in relatively low concentrations. The detection and analysis of these low-level proteins is hindered by the presence of a few proteins that are expressed in relatively high concentrations. In the case of muscle tissue, highly abundant structural proteins, such as actin, myosin, and tropomyosin, compromise the detection and analysis of more biologically relevant proteins. We have developed a practical protocol which exploits high-pH extraction to reduce or remove abundant structural proteins from skeletal muscle crude membrane preparations in a manner suitable for two dimensional gel electrophoresis. An initial whole-cell muscle lysate is generated by homogenization of powdered tissue in Tris-base. This lysate is subsequently partitioned into a supernatant and pellet containing the majority of structural proteins. Treatment of the pellet with high-pH conditions effectively releases structural proteins from membrane compartments which are then removed through ultracentrifugation. Mass spectrometric identification shows that the majority of protein spots reduced or removed by high-pH treatment were contractile proteins or contractile-related proteins. Removal of these proteins enabled successful detection and identification of minor proteins. Structural protein removal also results in significant improvement of gel quality and the ability to load higher amounts of total protein for the detection of lower abundant protein classes.     

2-D difference gel electrophoresis of the lung squamous cell carcinoma versus normal sera demonstrates consistent alterations in the levels of ten specific proteins

Most lung cancers are diagnosed too late for curative treatment to be possible, therefore early detection is crucial. Serum proteins are a rich source of biomarkers and have the potential to be used as diagnostic and prognostic indicators for lung cancer. In order to examine differences in serum levels of specific proteins associated with human lung squamous carcinoma, immunodepletion of albumin and five other high-abundant serum proteins followed by 2-D difference gel electrophoresis (DIGE) analysis and subsequent MS was used to generate a panel of proteins found to be differentially expressed between the cancer and normal samples. Proteins found to have increased abundance levels in squamous cell carcinoma sera compared to normal sera included apolipoprotein A-IV precursor, chain F; human complement component C3c, haptoglobin, serum amyloid A protein precursor and Ras-related protein Rab-7b. Proteins found to have lower abundance levels in squamous cell carcinoma sera compared to normal sera included alpha-2-HS glycoprotein, hemopexin precursor, proapolipoprotein, antithrombin III and SP40; 40. The data presented here demonstrate that high-abundant protein removal combined with 2-D DIGE is a powerful strategy for the discovery of potential biomarkers. The identification of lung cancer-specific biomarkers is crucial to early detection, which in turn could lead to a dramatic increase in survival rates.     

Sample preparation of human serum for the analysis of tumor markers. Comparison of different approaches for albumin and gamma-globulin depletion

LC-MS is a powerful method for the sensitive detection of proteins and peptides in biological fluids. However, the presence of highly abundant proteins often masks those of lower abundance and thus generally prevents their detection and identification in proteomic studies. In human serum the most abundant proteins are albumin and gamma-globulins. We tested several approaches to specifically reduce the level of these proteins based on either specific antibodies, dye ligands (for albumin) and protein A or G (for gamma-globulins). The resulting, depleted serum was analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis and LC-MS for the residual presence of these abundant proteins as well as for other serum proteins that should remain after depletion. To test the applicability of this method to real-life samples, depleted serum of a cervical cancer patient was analyzed for the presence of a specific tumor marker protein SCCA1 (squamous cell carcinoma antigen 1; P29508), which is present at ng/ml concentrations. The results demonstrate that SCCA1 can be detected by LC-MS in patient serum following depletion of albumin and gamma-globulins thus opening the possibility of screening patient sera for other, so far unknown, tumor markers.