Combinatorial Peptide Ligand Library Treatment Followed by a Dual-Enzyme, Dual-Activation Approach on a Nanoflow Liquid Chromatography/Orbitrap/Electron Transfer Dissociation System for Comprehensive Analysis of Swine Plasma Proteome

摘要: The plasma proteome holds enormous clinical potential, yet an in-depth analysis of the remains a daunting challenge due to its high complexity and extremely wide dynamic range in protein concentrations. Furthermore, existing antibody-based approaches for depleting high-abundance proteins are not adaptable animal proteome, which is often essential experimental pathology/pharmacology. Here we describe highly comprehensive method investigation employs optimized combinatorial peptide ligand library (CPLL) treatment reduce concentration dual-enzyme, dual-activation strategy achieve proteomic coverage. CPLL enriched lower abundance by >100-fold when samples were loaded moderately denaturing conditions with multiple loading-washing cycles. native CPLL-treated digested parallel two enzymes (trypsin GluC) carrying orthogonal specificities. By performing this differential proteolysis, coverage improved where peptides produced only one enzyme poorly detectable. Digests fractionated high-resolution strong cation exchange chromatography then resolved on long, heated nano liquid column. MS was performed linear triple quadrupole/orbitrap complementary activation methods (collisionally induced dissociation (CID) electron transfer dissociation). We applied investigate from swine, prominent model cardiovascular diseases (CVDs). This large-scale results identification total 3421 unique proteins, spanning 9-10 orders magnitude. identified under set commonly accepted criteria, including precursor mass error <15 ppm, Xcorr cutoffs, ≥2 at probability ≥95% ≥99%, false-positive rate data 1.8% as estimated searching reversed database. resulted 55% more over those plasma; moreover, compared using trypsin CID, dual-enzyme/activation approach enabled 2.6-fold substantially higher sequence most individual proteins. Further revealed 657 significantly associated CVDs (p < 0.05), constitute five CVD-related pathways. study represents first nonhuman developed here other complex proteomes.