We first evaluated our ability to solubilize proteins and high Mr
glycoconjugates from rat VF mucosa samples. illustrates representative 1-DE separation and positive immunoblotting of rat VF mucosa for the glycoprotein fibronectin and proteoglycan fibromodulin, confirming successful extraction and retention of these glycosylated ECM constituents. Fibronectin was detected at an expected 220×103 Mr
(native fibronectin is comprised of two 220×103 Mr
subunits which are separated on reducing SDS-PAGE) and appeared as a diffuse band suggesting varying degrees of glycosylation. Fibromodulin was detected as two distinct bands at 42 and 67×103 Mr
. Based on previous electrophoretic characterization 
, the 42×103 Mr
band is consistent with the non-glycosylated fibromodulin core protein and the 67×103 Mr
band is consistent with its N-linked oligosaccharide-substituted form. We did not observe evidence for a keratan sulfate-substituted form (typically detected as a series of diffuse bands between 70 and 110×103 Mr
) in these samples.
1-DE of rat vocal fold mucosa demonstrates high sample complexity and ECM glycoprotein/proteoglycan retention.
Next, we performed parallel LC-MS/MS runs on three independent samples, following initial Mr-based sample fractionation using 1-DE. Peptide and protein identifications were compared across independent sample runs in an attempt to salvage and validate potentially important low abundance proteins, as follows. Cross-sample matching was performed with special consideration of proteins identified by a single unique peptide. Proteins identified by a single unique peptide in a given sample (termed local single peptide hits) were categorized into two subsets: Those with a corresponding protein match in another sample (such a cross-sample match could have any number of peptide hits), and those with no corresponding protein match in another sample (termed global single peptide hits). Matching of protein identifications across samples was then performed with all peptide hits retained, with local single peptide hits removed, and with global single peptide hits removed.
We initially identified a total of 756 unique peptides associated with 340 proteins across all three samples, using a 1% estimated false discovery rate (). This analysis was marked by a significant number of local single peptide hits (108 [46.9% of 230] in sample 1; 98 [56.3% of 174] in sample 2; 119 [57.8% of 206] in sample 3). Removing all local single peptide hits prior to matching resulted in a 53.2% decrease in total proteins identified to 159, whereas removing only global single peptide hits resulted in a 37.9% decrease in total proteins identified to 211 (). Further, as the removal of global single peptide hits only affected protein identifications with no cross-sample matches, this strategy yielded improved percentage agreement across samples, resulting in 82.5% of identified proteins matched across at least two of three samples ().
Comparison of peptide and protein identifications generated from LC-MS/MS runs representing three independent vocal fold mucosa samples.
Detailed analysis of local single peptide hits () revealed that 30.6–46.3% of these protein identifications were global single peptide hits, confirming that the majority of protein identifications associated with a single unique peptide had a positive cross-sample match. Further, 19.4-39.8% of these identifications were matched across all three samples (). A large number of cross-sample matches were to other single unique peptides; however, some matches had as many as seven unique peptides (). To complement this analysis, we implemented secondary validation of MS/MS spectra associated with local single peptide hits using de novo
peptide sequencing followed by MS-driven BLAST searching 
. Thirty-two database hits failed this validation step and were therefore considered false positives.
Analysis of proteins identified by a single unique peptide in LC-MS/MS runs representing three independent vocal fold mucosa samples.
Table S1 contains functional classification data for proteins identified by LC-MS/MS following the removal of global single peptide hits and local single peptide hits derived from spectra that failed de novo
sequencing-based validation. Proteins were classified using annotation and categorization data in the UniProtKB/Swiss-Prot database 
. A wide range of cellular and extracellular proteins were identified, spanning 12 functional categories: Circulatory system, blood proteins; cytoskeletal proteins (microfilament, intermediate filament, microtubules) including nuclear envelope and epithelial keratins; DNA binding proteins; defense, stress and immune response proteins; ECM proteins; membrane (cell, nuclear, mitochondrial) proteins; metabolism and energy proteins; cell motility, contractile/thick filament proteins; protein fate (maturation, modification, trafficking, degradation); signaling proteins; protein translation/synthesis; and miscellaneous proteins.
We selected four representative VF mucosa proteins from Table S1 for additional immunohistochemical validation. The ECM protein collagen type I and glycoprotein fibronectin were detected throughout the LP, with preferential localization to the superficial LP (). The intermediate filament protein vimentin was detected in the cytosol of the majority of cells in the LP (); whereas the intermediate filament protein keratin Ka10 was exclusively localized to the epithelium ().
Representative images showing immunohistochemical validation of four vocal fold mucosa proteins identified using LC-MS/MS.