This approach, which has been employed elsewhere in tissue proteomics, would also allow accurate separation of the VF epithelium and LP, in addition to the investigation of regional areas of interest within the LP, such as the maculae flavae. The tightly regulated protein/Ponatinib glycan constituency of the LP ECM is critical to the biomechanical capacity of the VF mucosa for self-sustained oscillation. In this study, we successfully extracted and identified a number of procollagen/collagen isoforms, in addition to the proteoglycans decorin and fibromodulin, and the glycoproteins fibronectin, fibrillin and laminin. It is important to note that a number of known LP ECM constituents were not detected in our LC-MS/MS runs. As noted, ECM is generally a challenging target for proteomic analysis due to the high Mr, poor solubility and poor digestability of the majority of ECM proteins, many of which are extensively crosslinked and/or glycosylated. High Mr glycans and glycoconjugates are also known to impair isoelectric focusing during 2-DE. Work in other ECM-rich tissues such as cartilage has shown improved protein resolution on 2-DE following depletion of high Mr glycans using centrifugal filtration, anion exchange chromatography and cetylpyridinium chloride precipitation. Also, trypsin digestion of ECM prior to LC-MS/MS appears to be significantly enhanced by ultrasonication and incorporation of an acid-labile surfactant treatment. Analysis of ECM glycans and glycoconjugates may be best achieved by initial isolation from the larger proteome using antibody or lectin affinity chromatography, and/or metabolic labeling. Finally, compared to collision induced dissociation, electron transfer dissociation-based MS may be favorable for determining glycosylation site and glycan size, due its tendency to preferentially fragment the protein backbone while leaving glycan side chains largely intact. The validation of borderline protein identifications using crosssample matching of local single peptide hits in our dataset illustrates the value of performing MS/MS on multiple independent samples, and is a computationally straightforward approach to enhancing the identification of low abundance proteins. Further, secondary validation using de novo interpretation of relevant spectra provides additional protection against unwanted false positives. Conservative approaches to database-driven proteomics typically define a positive protein identification as characterized by two or more unique peptides. Although this approach stringently guards against false positives, it also removes a large number of potentially valuable protein identifications. The ideal management of single peptide hits involves maximizing true positive protein identifications while maintaining a strict false discovery rate. A recent body of literature in this area.