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 micro-environment via molecular and cellular profiles as tumor progresses would be vital for identifying cell or protein targets for cancer prevention and therapy.

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.

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: non-tumorigenic 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. Prot. Res. 5, 899-906 2006). The search files produced from 5 analyses (3 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 non-classical secretion (SecretomeIP, 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 and 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.

). † These authors contributed equally to this work. SummaryDue 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 14 N/ 15 N isotope coding strategy. After 2 months of growth on 14 N-and 15 N-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-

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.

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.

:  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.

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.