Hexapeptide Libraries for Enhanced Protein PTM Identification and Relative Abundance Profiling in Whole Human Saliva

Abstract

SUMMARY Dynamic range compression (DRC) by hexapeptide libraries increases MS/MS-based identification of lower-abundance proteins in complex mixtures. However, two unanswered questions impede fully realizing DRC’s potential in shotgun proteomics. First, does DRC enhance identification of post-translationally modified proteins? Second, can DRC be incorporated into a workflow enabling relative protein abundance profiling? We sought to answer both questions analyzing human whole saliva. Addressing question one, we coupled DRC with covalent glycopeptide enrichment and MS/MS. With DRC we identified ~2 times more N-linked glycoproteins and their glycosylation sites than without DRC, dramatically increasing the known salivary glycoprotein catalog. Addressing question two, we compared differentially stable isotope-labeled saliva samples pooled from healthy and metastatic breast cancer women using a multidimensional peptide fractionation-based workflow, analyzing in parallel one sample portion with DRC and one portion without. Our workflow categorizes proteins with higher absolute abundance, whose relative abundance ratios are altered by DRC, from proteins of lower absolute abundance detected only after DRC. Within each of these salivary protein categories we identified novel abundance changes putatively associated with breast cancer, demonstrating feasibility and benefits of DRC for relative abundance profiling. Collectively, our results bring us closer to realizing the full potential of DRC for proteomic studies.

“Cheek-to-cheek” urinary proteome profiling via combinatorial peptide ligand libraries: A novel, unexpected elution system

“…The latter thus occurs mostly via ionic and hydrogen bonds, bonds that, in turn, are easily challenged by salts, especially positively charged ones such as Tris and free Lys (see Figs. [2][3][4][5]. As a proof of that is the fact that, in the presence of such high levels of adsorption of bile acids and other pigments, the binding of albumin to ProteoMiner is severely quenched.…”

The human saliva proteome: overview and emerging methods for characterization

“…Quantitative protein profiling is also done routinely. Quantitative studies have identified salivary proteins associated with oral cancer 11 , 12 and also systemic conditions such as 2 diabetes 13 and breast cancer 14,15 .…”

“…Post-translation modifications (PTMs), which regulated structure and function of proteins, are possibly the most important aspect of protein biochemistry that can be characterized via MS. In saliva, a number of studies have targeted glycosylation 19,20 , generating a catalog of hundreds of proteins in saliva carrying N-linked glycosylation modifications 21,15 . The phosphoproteome has also been characterized in saliva, with one study identifying phosphorylation events on proline-rich proteins 22 .…”

Human saliva proteome: an overview

“…Multiple groups have quantitatively characterized the whole saliva proteome to determine proteins associated with oral cancer 11 , 12 . Others have quantitatively profiled saliva for proteins associated with systemic health conditions, such as type-2 diabetes 13 and breast cancer 14,15 .…”

“…In an extracellular fluid like saliva, glycosylation is a prominent protein PTM, providing critical functions to proteins 19,20 . Several studies have used biochemical enrichment of glycosylated proteins, followed by shotgun MS/MS to catalog these proteins in soluble whole saliva 21,15 resulting in a catalog of hundreds of proteins known to carry glycosylation PTMs.…”