A Straightforward and Highly Efficient Precipitation/On-Pellet Digestion Procedure Coupled with a Long Gradient Nano-LC Separation and Orbitrap Mass Spectrometry for Label-Free Expression Profiling of the Swine Heart Mitochondrial Proteome

Abstract

For label-free expression profiling of tissue proteomes, efficient protein extraction, thorough and quantitative sample cleanup and digestion procedures, as well as sufficient and reproducible chromatographic separation, are highly desirable but remain challenging. However, optimal methodology has remained elusive, especially for proteomes that are rich in membrane proteins, such as the mitochondria. Here we describe a straightforward and reproducible sample preparation procedure, coupled with a highly selective and sensitive nano-LC/Orbitrap analysis, which enables reliable and comprehensive expression profiling of tissue mitochondria. The mitochondrial proteome of swine heart was selected as a test system. Efficient protein extraction was accomplished using a strong buffer containing both ionic and non-ionic detergents. Overnight precipitation was used for cleanup of the extract, and the sample was subjected to an optimized 2-step, on-pellet digestion approach. In the first step, the protein pellet was dissolved via a 4 h tryptic digestion under vigorous agitation, which nano-LC/LTQ/ETD showed to produce large and incompletely cleaved tryptic peptides. The mixture was then reduced, alkylated, and digested into its full complement of tryptic peptides with additional trypsin. This solvent precipitation/on-pellet digestion procedure achieved significantly higher and more reproducible peptide recovery of the mitochondrial preparation, than observed using a prevalent alternative procedure for label-free expression profiling, SDS-PAGE/ingel digestion (87% vs. 54%). Furthermore, uneven peptide losses were lower than observed with SDS-PAGE/in-gel digestion. The resulting peptides were sufficiently resolved by a 5 h gradient using a nano-LC configuration that features a low-void-volume, high chromatographic reproducibility, and an LTQ/Orbitrap analyzer for protein identification and quantification. The developed method was employed for label-free comparison of the mitochondrial proteomes of myocardium from healthy animals vs. those with hibernating myocardium. Each experimental group consisted of a relatively large number of animals (n=10), and samples were analyzed in random order to minimize quantitative false-positives. Using this approach, 904 proteins were identified and quantified with high confidence, and those mitochondrial proteins that were altered significantly between groups were

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

“…The protein concentration was determined with the BCA protein assay after an ultracentrifugation (140,000 ϫ g, 40 min, 4°C). To remove undesirable components while maintaining a high peptide recovery, we employed a precipitation/on-pellet-digestion protocol that we developed previously (22) for the digestion with either trypsin or GluC. For GluC digestion, the RapGest cleavable detergent was added to a concentration of 0.1% to facilitate pellet dissolution.…”

Cisplatin-induced Ototoxicity Is Mediated by Nitroxidative Modification of Cochlear Proteins Characterized by Nitration of Lmo4

“…The details for sample preparation and dual-activation nano-LC/MS analysis are described in previous publications (26 -28). Briefly, a precipitation of the sample was digested after an onpellet-digestion protocol (26), where a dynamic flow nanospray interface was used to clean and digest the samples. A high resolution nano-LC separation was employed to resolve the tryptic peptides.…”

Regulation of Polyhydroxybutyrate Accumulation in Sinorhizobium meliloti by the Trans -Encoded Small RNA MmgR

“…The proteins from both fractions were precipitated overnight with 6 volumes of ice-cold acetone and stored as dry pellets. An on-pellet trypsin digestion was performed after pellet resuspension in 50 mM Tris-HCl (pH 8.5) buffer, followed by peptide reduction with dithiothreitol and alkylation with iodoacetamide, as described previously (66). The mass spectrometric analysis of the samples was performed with an Orbitrap Velos Pro mass spectrometer (Thermo Scientific, Germany).…”