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

Abstract

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

Proteomic Analysis Reveals Diverse Classes of Arginine Methylproteins in Mitochondria of Trypanosomes

“…As a result, combining a high-resolution MS analyzer with alternating CID and ETD analyses enabled a comprehensive characterization of arg methylated peptides/proteins (26). More recently (20,46), we demonstrated that the dual-activation method, in conjunction with proteolysis with orthogonal enzymes (e.g. trypsin and gluC) and efficient chromatographic separations, is capable of achieving a highly comprehensive analysis of proteins and post-translational modifications in very complex proteomes.…”

Phosphorylation of Tyr188 in the WW domain of YAP1 plays an essential role in YAP1-induced cellular transformation

“…The band containing YAP1 were excised and digested with in-gel digestion protocol with trypsin only and solution samples were divided into 2 aliquots for dual enzyme digestion of trypsin and GluC according to the 2-step on pellet digestion protocol. 22,23 The digests were eluted on a 100 cm column with a 2-hr gradient with CID and ETD activation by Fusion Tribrid mass spectrometer (Thermo Scientific), using a previously described condition. 24 Raw files were searched with Proteome Discoverer (Thermo Scientific) against YAP1 sequence and Carbamidomethyl (57.021Da) was set as static modification on Cystine (C), and oxidation (15.995Da ) on methionine (M); phosphorylation (79.966 Da) on serine (S), threonine (T) and tyrosine (Y) were set as dynamic side chain modifications.…”

Mark Twain: How to fathom the depth of your pet proteome

“…Fig. 3, from Tu et al [30] tells a tall tale: when using plain, unfractionated plasma, the lowest detection limit appears to be 10 ng/mL. However, if plasma is pre-treated with ProteoMiner, species can be detected down to as low as 10 pg protein per mL, with an increment of sensitivity of up to three orders of magnitude.…”

“…Note how, in the first case, the lower limit of detection is 10 ng/mL whereas after CPLL capture the lowest sensitivity limit is 10 pg/mL. Modified from Tu et al [30] where details can be found, by permission.…”