Blood plasma is the most complex human-derived proteome, containing other tissue proteomes as subsets. This proteome has only been partially characterized due to the extremely wide dynamic range of the plasma proteins of more than ten orders of magnitude. Thus, the reduction in sample complexity prior to mass spectrometric analysis is particularly important and alternative separation methodologies are required to more effectively mine the lower abundant plasma proteins. Here, we demonstrated a novel separation approach using 2-D free-flow electrophoresis (FFE) separating proteins and peptides in solution according to their pI prior to LC-MS/MS. We used the combination of sequential protein and peptide separation by first separating the plasma proteins into specific FFE fractions. Tryptic digests of the separated proteins were generated and subsequently separated using FFE. The protein separation medium was optimized to segregate albumin into specific fractions containing only few other proteins. An optimization of throughput for the protein separation reduced the separation time of 1 mL of plasma to approximately 3 h providing sufficient material for digestion and the subsequent peptide separation. Our approach revealed low-abundant proteins (e.g., L-selectin at 17 ng/mL and vascular endothelial-cadherin precursor at 30 ng/mL) and several tissue leakage products, thus providing a powerful orthogonal separation step in the proteomics workflow.
We present a new procedure for in-gel digestion of proteins introducing a combination of two different 96-well microplates. The two plates have incorporated small capillaries with a length of 2.4 mm in each well, one of which has 75-microm-inner diameter capillaries, whereas the second plate has reversed-phase-type capillaries fixed to it. The initial steps of the in-gel digestion process, comprising destaining, reduction/alkylation, dehydration, and digestion, was carried out in the plate containing 75-microm capillaries. Capillaries containing C18 reversed-phase modified monolithic silica rods of a 200-microm diameter were used for the second plate in which extraction and cleanup of peptides were carried out. Peptides were eluted directly from the solid-phase extraction plate onto the MALDI sample support. The separation of the process into two plates led to increased process stability, without compromising sensitivity, i.e. peptide recovery, making it suitable for true high-throughput protein identification. The handling of proteinases could easily be optimized, and no restrictions were made on chosen pH range through the absence of the solid phase in the initial steps of the protocol. Efficient binding of peptides to the solid phase and subsequent direct elution onto the MALDI sample support led to sensitivities in the attomole range. Performance of the process was demonstrated with tryptic digests of proteins stained with colloidal coomassie blue, silver, and the fluorescent stain SYPRO Ruby.
No abstract
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.