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Abstract Background Activated leukocyte cell adhesion molecule (ALCAM) is implicated in the prognosis of multiple cancers with low level expression associated with metastasis and early death in breast cancer. Despite this significance, mechanisms that regulate ALCAM gene expression and ALCAM's role in adhesion of pre-metastatic circulating tumor cells have not been defined. We studied ALCAM expression in 20 tumor cell lines by real-time PCR, western blot and immunochemistry. Epigenetic alterations of the ALCAM promoter were assessed using methylation-specific PCR and bisulfite sequencing. ALCAM's role in adhesion of tumor cells to the vascular wall was studied in isolated perfused lungs. Results A common site for transcription initiation of the ALCAM gene was identified and the ALCAM promoter sequenced. The promoter contains multiple cis-active elements including a functional p65 NF-κB motif, and it harbors an extensive array of CpG residues highly methylated exclusively in ALCAM-negative tumor cells. These CpG residues were modestly demethylated after 5-aza-2-deoxycytidine treatment. Restoration of high-level ALCAM expression using an ALCAM cDNA increased clustering of MDA-MB-435 tumor cells perfused through the pulmonary vasculature of ventilated rat lungs. Anti-ALCAM antibodies reduced the number of intravascular tumor cell clusters. Conclusion Our data suggests that loss of ALCAM expression, due in part to DNA methylation of extensive segments of the promoter, significantly impairs the ability of circulating tumor cells to adhere to each other, and may therefore promote metastasis. These findings offer insight into the mechanisms for down-regulation of ALCAM gene expression in tumor cells, and for the positive prognostic value of high-level ALCAM in breast cancer.

Context: Eryngium foetidum L. (Apiaceae) is a traditional herb that has been used for numerous medicinal applications, including as a treatment for parasitic infections, especially in the Neotropics from where it originates., Objective: This study evaluates the in vitro leishmanicidal and cytotoxicity activities of isolated compounds based on a bioassay-guided fractionation approach., Materials and Methods: Defatted aerial parts of E. foetidum were subjected to extraction with methanol followed by partitioning with n-hexane, ethyl acetate and 50% methanol. Then, the first two fractions were subsequently fractionated by column chromatography and HPLC. Compound identity was confirmed by mass spectrometry and NMR spectroscopy. Leishmania tarentolae (promastigotes) and L. donovani (amastigotes) were used as testing parasites. L6 rat myoblasts were used for cytotoxicity. All extracts and fractions were tested at 20 μg/mL., Results: The initial methanol extract showed 20% growth inhibition of L. tarentolae. Then, the n-hexane and ethyl acetate fractions were also active showing approximately 40% growth inhibition. From these two fractions, the following compounds were isolated: lasidiol p-methoxybenzoate (1), a daucane sesquiterpene; and 4-hydroxy-1,1,5-trimethyl-2-formyl-cyclohexadien-(2,5)-[α-acetoxymethyl-cis-crotonate] (2), a terpene aldehyde ester derivative. Compound 1 inhibited the growth of both L. tarentolae and L. donovani with IC₅₀ values of 14.33 and 7.84 μM, respectively; and showed no cytotoxicity (IC₅₀ > 50 μM). Compound 2 was inactive in the L. tarentolae assay (IC₅₀ > 50 μM)., Discussion and Conclusion: This study presented the bioassay-guided fractionation with the leishmanicidal and cytotoxicity activities of two compounds isolated for the first time from an Eryngium species.

Anthocyanins and phenolic acids are major secondary metabolites in blueberry with important implications for human health maintenance. An improved protocol was developed for the accurate, efficient, and rapid comparative screening for large blueberry sample sets. Triplicates of six commercial cultivars and four breeding selections were analyzed using the new method. The compound recoveries ranged from 94.2 to 97.5 ± 5.3% when samples were spiked with commercial standards prior to extraction. Eighteen anthocyanins and 4 phenolic acids were quantified in frozen and freeze-dried fruits. Large variations for individual and total anthocyanins, ranging from 201.4 to 402.8 mg/100 g, were assayed in frozen fruits. The total phenolic acid content ranged from 23.6 to 61.7 mg/100 g in frozen fruits. Across all genotypes, freeze-drying resulted in minor reductions in anthocyanin concentration (3.9%) compared to anthocyanins in frozen fruits. However, phenolic acids increased by an average of 1.9-fold (±0.3) in the freeze-dried fruit. Different genotypes frequently had comparable overall levels of total anthocyanins and phenolic acids, but differed dramatically in individual profiles of compounds. Three of the genotypes contained markedly higher concentrations of delphinidin 3-O-glucoside, cyanidin 3-O-glucoside, and malvidin 3-O-glucoside, which have previously been implicated as bioactive principles in this fruit. The implications of these findings for human health benefits are discussed.

Unlabelled: The human health benefits from consumption of cranberry products have been associated with the fruits' unique flavonoid composition, including a complex profile of anthocyanins and proanthocyanidins. However, when processed by techniques such as pressing, canning, concentrating, or drying, a number of these natural components may be compromised or inactivated due to physical separation, thermal degradation, or oxidation. Fresh cranberries were compared to freeze-dried berries and individual fruit tissues (skin and peeled fruit). Products examined included cranberry juices (commercial and prepared from concentrate), cranberry sauces (commercial and homemade), and sweetened-dried cranberries (commercial). Freeze-drying resulted in no detectable losses of anthocyanins or proanthocyanidins from cranberry fruits. Anthocyanins were localized in the skin. Proanthocyanins were higher in the skin than in the flesh, with the exception of procyanidin A-2 dimer which was concentrated in the flesh. Anthocyanins were significantly higher in not-from-concentrate juice than in reconstituted juice from concentrate (8.3 mg and 4.2 mg/100 mL, respectively). Similarly, proanthocyanidins were markedly higher in not-from-concentrate juice compared to juice from concentrate (23.0 mg and 8.9 mg/100 mL, respectively). Homemade sauce contained far higher anthocyanins and proanthocyanidins (15.9 and 87.9 mg/100 g, respectively) than canned sauces processed with whole berries (9.6 and 54.4 mg/100 g, respectively) or jelled-type (1.1 and 16 mg/100 g, respectively). Sweetened-dried cranberries were quite low in anthocyanins (7.9 mg/100 g), but they still retained considerable proanthocyanidins (64.2 mg/100 g). Commercially processed products contained significantly lower levels of polyphenols as compared to fresh and home-processed preparations. Anthocyanins were more sensitive to degradation than proanthocyanidins., Practical Application: As cranberry juices and other products are increasingly consumed for their recognized health benefits (including prophylaxis against urinary tract infection), it is relevant to consider how various degrees of commercial and home processing can alter innate levels of the biologically active flavonoids (especially anthocyanins and proanthocyanidins) characteristic to the intact fruits., (© 2012 Institute of Food Technologists®)

Blackcurrant is considered as a natural high-value food raw material and possesses a variety of therapeutic properties. The health benefits of blackcurrant have generally been credited to its high anthocyanin content; however, the therapeutic properties of other minor flavonoids constituents have not yet been investigated due the difficulties related to their isolation. Multiple steps of high-performance counter-current chromatography in combination with ESI tandem mass spectrometry (MS(n)) were successfully used for the preparative isolation of flavonols from blackcurrant extract, to study their electrospray ionization mass spectrometry fragmentation behavior. Seven flavonols, namely myricetin-3-O-rutinoside (145.5 mg), myricetin-3-O-hexoside (79.7 mg), myricetin-3-O-(6″-malonyl)-glucoside (17.4 mg), kaempferol-3-O-glucoside (20.5 mg), quercetin-3-O-rutinoside (55.1 mg), quercetin-3-O-hexoside (25.8 mg), and myricetin (129.1 mg) have been successfully isolated and their multistage MS(n) data were used for detailed structure characterization. The results of these experiments demonstrated that high-performance counter-current chromatography along with ESI-MS(n) is a sensitive, selective, and effective technology for isolation and characterization of minor constituents from a complex mixture., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Alkaloids from the stem bark of Geissospermum vellosii possess a variety of therapeutic properties including antimalarial activities, activity as a sexual stimulant and inhibition of the proliferation of HIV and herpes viruses. Methods currently used to isolate the active components from G. vellosii are time-consuming, labor intensive, and result in low recovery. In addition, there is a lack of sensitive and accurate analytical methods for the structural characterization and identification of alkaloid components in minor quantities. A combination of high performance counter-current chromatography and ESI tandem mass spectrometry (MS(n)) was established to isolate alkaloids from the stem bark of G. vellosii, and study their electrospray ionization mass spectrometry fragmentation behavior. Five indole alkaloids were successfully isolated and identified by nuclear magnetic resonance and mass spectrometry. The multi-stage tandem mass spectrometric data were used to study their fragmentation pattern and set a model for detailed structure characterization of related indole alkaloids. The presence of the even mass fragment ion suggestive of an odd number of nitrogen at m/z 144 corresponding to C(10)H(9)N was characteristic to indole alkaloids. The results of the experiments demonstrated that the combination of high performance counter current chromatography and ESI-MS(n) is a sensitive, selective and effective approach for rapid isolation and characterization of alkaloids from G. vellosii., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Ethnopharmacological Relevance: The stem bark of Geissospermum vellosii has been traditionally used by the native population of northern South America to treat malaria. Indole alkaloids have been previously isolated from this plant, but the antiplasmodial constituents have not yet been described. As part of our ongoing investigations of new bioactive compounds with activity against malaria parasites, we tested the in vitro antiplasmodial activity of isolated fractions and purified alkaloids from Geissospermum vellosii., Materials and Methods: Indole alkaloids were isolated and identified from a methanolic crude extract of Geissospermum vellosii bark using a combination of high performance counter current chromatography, mass spectrometry and nuclear magnetic resonance technologies. The methanolic extract, the crude alkaloid fractions and the purified compounds were tested for in vitro antiplasmodial activity against the chloroquine-sensitive strain of Plasmodium falciparum (D10)., Results: An indole alkaloid (4) along with four known indole alkaloids, geissolosimine (1), geissospermine (2), geissoschizoline (3), and vellosiminol (5) were isolated and structure elucidated. The antiplasmodial activity (IC(50)) of the methanolic crude extract was 2.22 μg/mL, while for the isolated compounds it ranged from 0.96 μM to 13.96 μM except for (5) which showed a low activity (157 μM). Geissolosimine (1) showed the highest antiplasmodial activity (0.96 μM)., Conclusions: This study provides evidence to support the use of Geissospermum vellosii as an antimalarial agent, as used by the native populations. Geissolosimine (1) is a lead molecular structure for possible antimalarial drug development., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

The development of new antiplasmodial drugs is of primary importance due to the growing problem of multi-drug resistance of malaria parasites. Spilanthes acmella, a plant traditionally used for the treatment of toothache, was targeted as a lead for its potential antiplasmodial activity. A systematic approach for investigating a suitable centrifugal partition chromatography (CPC) solvent system for N-alkylamides separation was reported. The partition behavior of three N-alkylamides has been studied using several biphasic solvent mixtures in search of an adequate CPC solvent system for this class of compounds. Major N-alkylamides in S. acmella were isolated from a methanolic crude extract of flowers by CPC with the solvent system heptanes-ethyl acetate-methanol-water (3:2:3:2, v/v/v/v). Four N-alkylamides were purified and the structures were illustrated by electrospray ionization-ion trap-time of flight-mass spectrometry (ESI-IT-TOF-MS), ¹H nuclear magnetic resonance (¹H NMR) and ¹³C nuclear magnetic resonance (¹³C NMR). The CPC fractions, which contained natural mixtures of phytochemicals, demonstrated significantly higher antiplasmodial activity compared to corresponding purified N-alkylamides, thus suggesting that interactions between these N-alkylamides may potentiate antiplasmodial bioactivity., (Copyright © 2011 Elsevier B.V. All rights reserved.)

To evaluate the implementation of various denaturants and their efficacy in bottom-up membrane proteomic methods using LC-MS analysis, microsomes isolated from tomato roots were treated with MS-compatible surfactants (RapiGest SF Surfactant from Waters and PPS Silent Surfactant from Protein Discovery), a chaotropic reagent (guanidine hydrochloride), and an organic solvent (methanol). Peptides were analyzed in triplicate sample and technical replicates by data-independent LC-MS(E) analysis. Overall, 2333 unique peptides matching to 662 unique proteins were detected with the order of denaturant method efficacy being RapiGest SF Surfactant, PPS Silent Surfactant, guanidine hydrochloride, and methanol. Using bioinformatic analysis, 103 proteins were determined to be integral membrane proteins. When normalizing the data as a percentage of the overall number of peptides and proteins identified for each method, the order for integral membrane protein identification efficacy was methanol, guanidine hydrochloride, RapiGest SF Surfactant, and PPS Silent Surfactant. Interestingly, only 8% of the proteins were identified in all four methods with the silent surfactants having the greatest overlap at 17%. GRAVY analysis at the protein and peptide level indicated that methanol and guanidine hydrochloride promoted detection of hydrophobic proteins and peptides, respectively; however, trypsin activity in the presence of each denaturant was determined as a major factor contributing to peptide identification by LC-MS(E) . These results reveal the complementary nature of each denaturant method, which can be used in an integrated approach to provide a more effective bottom-up analysis of membrane proteomes than can be achieved using only a single denaturant., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Activated leukocyte cell adhesion molecule (ALCAM/CD166) is expressed by hematopoietic stem cells. However, its role in hematopoietic differentiation has not previously been defined., Results: In this study, we show that ALCAM expression is silenced in erythromegakaryocytic progenitor cell lines. In agreement with this finding, the ALCAM promoter is occupied by GATA-1 in vivo, and a cognate motif at -850 inhibited promoter activity in K562 and MEG-01 cells. Gain-of-function studies showed that ALCAM clusters K562 cells in a process that requires PKC. Induction of megakaryocytic differentiation in K562 clones expressing ALCAM activated PKC-δ and triggered apoptosis., Conclusions: There is a lineage-specific silencing of ALCAM in bi-potential erythromegakaryocytic progenitor cell lines. Marked apoptosis of ALCAM-expressing K562 clones treated with PMA suggests that aberrant ALCAM expression in erythromegakaryocytic progenitors may contribute to megakaryocytopenia.

The hexane extract from the leaves of Canella winterana exhibited strong activity against the chloroquine sensitive (CQS) strain of Plasmodium falciparum (D10) in vitro (IC50 2.53 microg/mL). Bioassay guided fractionation of this extract has led to the isolation of 5 drimane-type sesquiterpenoids: 9-epideoxymuzigadial, 9-deoxymuzigadial, muzigadial, 3-beta-acetoxypolygodial and the newly isolated hemiacetal, named muzigodiol, with IC50-values of 1.01, 2.19, 0.31, 2.77 and 7.43 microg/mL, respectively. The first four compounds were tested for their cytotoxicity using Chinese Hamster Ovarian (CHO) cells, where they showed IC50-values of 1.82, 33.69, 1.18, and 58.31 microg/mL, respectively. A structure-activity relationship is discussed.

Background: Activated leukocyte cell adhesion molecule (ALCAM) is implicated in the prognosis of multiple cancers with low level expression associated with metastasis and early death in breast cancer. Despite this significance, mechanisms that regulate ALCAM gene expression and ALCAM's role in adhesion of pre-metastatic circulating tumor cells have not been defined. We studied ALCAM expression in 20 tumor cell lines by real-time PCR, western blot and immunochemistry. Epigenetic alterations of the ALCAM promoter were assessed using methylation-specific PCR and bisulfite sequencing. ALCAM's role in adhesion of tumor cells to the vascular wall was studied in isolated perfused lungs., Results: A common site for transcription initiation of the ALCAM gene was identified and the ALCAM promoter sequenced. The promoter contains multiple cis-active elements including a functional p65 NF-κB motif, and it harbors an extensive array of CpG residues highly methylated exclusively in ALCAM-negative tumor cells. These CpG residues were modestly demethylated after 5-aza-2-deoxycytidine treatment. Restoration of high-level ALCAM expression using an ALCAM cDNA increased clustering of MDA-MB-435 tumor cells perfused through the pulmonary vasculature of ventilated rat lungs. Anti-ALCAM antibodies reduced the number of intravascular tumor cell clusters., Conclusion: Our data suggests that loss of ALCAM expression, due in part to DNA methylation of extensive segments of the promoter, significantly impairs the ability of circulating tumor cells to adhere to each other, and may therefore promote metastasis. These findings offer insight into the mechanisms for down-regulation of ALCAM gene expression in tumor cells, and for the positive prognostic value of high-level ALCAM in breast cancer.

On the basis of the complete genome sequence of the root-knot nematode Melodogyne hapla, we have deduced and annotated the entire proteome of this plant-parasite to create a database of 14,420 proteins. We have made this database, termed HapPep3, available from the Superfamily repository of model organism proteomes (http://supfam.mrc-lmb.cam.ac.uk/SUPERFAMILY). To experimentally confirm the HapPep3 assignments using proteomics, we applied a data-independent LC/MS(E) analysis to M. hapla protein extracts fractionated by SDS-PAGE. A total of 516 nonredundant proteins were identified with an average of 9 unique peptides detected per protein. Some proteins, including examples with complex gene organization, were defined by more than 20 unique peptide matches, thus, providing experimental confirmation of computational predictions of intron/exon structures. On the basis of comparisons of the broad physicochemical properties of the experimental and computational proteomes, we conclude that the identified proteins reflect a true and unbiased sampling of HapPep3. Conversely, HapPep3 appears to broadly cover the protein space able to be experimentally sampled. To estimate the false discovery rate, we queried human, plant, and bacterial databases for matches to the LC/MS(E)-derived peptides, revealing fewer than 1% of matches, most of which were to highly conserved proteins. To provide a functional comparison of the acquired and deduced proteomes, each was subjected to higher order annotation, including comparisons of Gene Ontology, protein domains, signaling, and localization predictions, further indicating concordance, although those proteins that did deviate seem to be highly significant. Approximately 20% of the experimentally sampled proteome was predicted to be secreted, and thus potentially play a role at the host-parasite interface. We examined reference pathways to determine the extent of proteome similarity of M. hapla to that of the free-living nematode, Caenorhabditis elegans, revealing significant similarities and differences. Collectively, the analyzed protein set provides an initial foundation to experimentally dissect the basis of plant parasitism by M. hapla.

Performance differences in protein and proteome characterization achieved by data-independent acquisition (DIA) LC/MS(E) and data-dependent acquisition (DDA) LC/MS/MS approaches were investigated. LC/MS(E) is a novel mode of generating product ion data for all coeluting precursors in parallel as opposed to LC/MS/MS where coeluting precursors must be serially fragmented one at a time. During LC/MS(E) analysis, alternating MS scans of "normal" and "elevated" collision energy are collected at regular intervals, providing nearly a 100% duty cycle for precursor detection and fragmentation because all precursors are fragmented across their full chromatographic elution profile. This is in contrast to DDA-based MS/MS where serial selection of precursor ions is biased toward interrogation and detection of the highest abundance sample components by virtue of the intensity-driven interrogation scheme employed. Both modes of acquisition were applied to a simple four-protein standard mixture with a 16-fold dynamic range in concentration, an in-gel digest of the Arabidopsis thaliana protein FLS2 purified by immunoprecipitation, and a solution-digested tomato leaf proteome sample. Dramatic improvement for individual protein sequence coverage was obtained for all three samples analyzed by the DIA approach, particularly for the lowest abundance sample components. In many instances, precursors readily detected and identified during DIA were either interrogated by MS/MS during DDA at inopportune points in their chromatographic elution profiles resulting in poor quality product ion spectra or not interrogated at all. Detailed evaluation of both DDA and DIA raw data and timing of the MS-to-MS/MS switching events clearly revealed the fundamental limitations of serial MS/MS interrogation and the advantages of parallel fragmentation by DIA for more comprehensive protein identification and characterization which holds promise for enhanced isoform and post-translational modification analysis.

Purpose: The role of the microenvironment during the initiation and progression of carcinogenesis is now realized to be of critical importance, both for enhanced understanding of fundamental cancer biology, as well as exploiting this source of relatively new knowledge for improved molecular diagnostics and therapeutics., Methods: This review focuses on: (1) the approaches of preparing and analyzing secreted proteins, (2) the contribution of tumor microenvironment elements in cancer, and (3) the potential molecular targets for cancer therapy., Results: The microenvironment of a tumor is an integral part of its physiology, structure, and function. It is an essential aspect of the tumor proper, since it supplies a nurturing environment for the malignant process. A fundamental deranged relationship between tumor and stromal cells is essential for tumor cell growth, progression, and development of life threatening metastasis. Improved understanding of this interaction may provide new and valuable clinical targets for cancer management, as well as risk assessment and prevention. Non-malignant cells and secreted proteins from tumor and stromal cells are active participants in cancer progression., Conclusions: Monitoring the change in the tumor microenvironment via molecular and cellular profiles as tumor progresses would be vital for identifying cell or protein targets for cancer prevention and therapy.

Due to ease of manipulation, metabolic isotope coding of samples for proteomic analysis is typically performed in cell culture, thus preventing an accurate in vivo quantitative analysis, which is only achievable in intact organisms. To address this issue in plant biology, we developed SILIP (stable isotope labeling in planta) using tomato plants (Solanum lycopersicum cv. Rutgers) as a method that allows soil-grown plants to be efficiently labeled using a 14N/15N isotope coding strategy. After 2 months of growth on 14N- and 15N-enriched nitrogen sources, proteins were extracted from four distinct tomato tissues (roots, stems, leaves and flowers), digested, and analyzed by LC/MS/MS (data-dependent acquisition, DDA) and alternating low- and elevated-energy MS scans (data-independent acquisition, MS(E)). Using a derived relationship to generate a theoretical standard curve, the measured ratio of the M (monoisotopic) and M-1 isotopologues of 70 identified 15N-labeled peptides from 16 different proteins indicated that 15N incorporation was almost 99%, which is in excellent agreement with the 99.3% 15N-enriched nitrate used in the soil-based medium. Values for the various tissues ranged from 98.2 +/- 0.3% 15N incorporation in leaves to 98.8 2 +/- 0.2% in stems, demonstrating uniform labeling throughout the plant. In addition, SILIP is compatible with root-knot nematode (Meloidogyne spp.) development, and thus provides a new quantitative proteomics tool to study both plant and plant-microorganism systems.

Proteins secreted (the secretome) from cancer cells are potentially useful as biomarkers of the disease. Using LC-MS/MS, the secreted proteomes from a series of isogenic breast cancer cell lines varying in aggressiveness were analyzed by mass spectrometry: nontumorigenic MCF10A, premalignant/tumorigenic MCF10AT, tumorigenic/locally invasive MCF10 DCIS.com, and tumorigenic/metastatic MCF 10CA cl. D. Proteomes were obtained from conditioned serum-free media, partially fractionated using a small reverse phase C2 column, and digested with trypsin for analysis by LC-MS/MS, using a method previously shown to give highly enriched secreted proteomes (Mbeunkui et al. J. Proteome Res. 2006, 5, 899-906). The search files produced from five analyses (three separate preparations) were combined for database searching (Mascot) which produced a list of over 250 proteins from each cell line. The aim was to discover highly secreted proteins which changed significantly in abundance corresponding with aggressiveness. The most apparent changes were observed for alpha-1-antichymotrypsin and galectin-3-binding protein which were highly secreted proteins from MCF10 DCIS.com and MCF10CA cl. D, yet undetected in the MCF10A and MCF10AT cell lines. Other proteins showing increasing abundance in the more aggressive cell lines included alpha-1-antitrypsin, cathepsin D, and lysyl oxidase. The S100 proteins, often associated with metastasis, showed variable changes in abundance. While the cytosolic proteins were low (e.g., actin and tubulin), there was significant secretion of proteins often associated with the cytoplasm. These proteins were all predicted as products of nonclassical secretion (SecretomeP, Center for Biological Sequence Analysis). The LC-MS/MS results were verified for five selected proteins by western blot analysis, and the relevance of other significant proteins is discussed. Comparisons with two other aggressive breast cancer cell lines are included. The protein with consistent association with aggressiveness in all lines, and in unrelated cancer cells, was the galectin-3-binding protein which has been associated with breast, prostate, and colon cancer earlier, supporting the approach and findings. This analysis of an isogenic series of cell lines suggests the potential usefulness of the secretome for identifying prospective markers for the early detection and aggressiveness/progression of cancer.

Reliable methods for profiling secretory proteins are highly desirable for the identification of biomarkers of disease progression. Secreted proteins are often masked by high amounts of protein supplements in the culture medium. We have developed an efficient method for the enrichment and analysis of the secretome of different cancer cell lines, free of essential contaminants. The method is based on the optimization of cell incubation conditions in protein-free medium. Secreted proteins are concentrated and fractionated using a reversed-phase tC2 Sorbent, followed by peptide mass fingerprinting for protein identification. An average of 88 proteins were identified in each cancer cell line, of which more than 76% are known to be secreted, possess a signal peptide or a transmembrane domain. Given the importance of secreted proteins as a source for early detection and diagnosis of disease, this approach may help to discover novel candidate biomarkers with potential clinical significance.