Abstract
Multidimensional peptide separations can greatly increase the depth of coverage in proteome profiling. However, a major challenge for multidimensional separations is the requirement of large biological samples, often containing milligram amounts of protein. We have developed nanowell-mediated two-dimensional (2D) reversed-phase nanoflow liquid chromatography (LC) separations for in-depth proteome profiling of low-nanogram samples. Peptides are first separated using high-pH LC and the effluent is concatenated into 4 or 12 nanowells. The contents of each nanowell are reconstituted in LC buffer and collected for subsequent separation and analysis by low-pH nanoLC-MS/MS. The nanowell platform minimizes peptide losses to surfaces in offline 2D LC fractionation, enabling >5800 proteins to be confidently identified from just 50 ng of HeLa digest. Furthermore, in combination with a recently developed nanowell-based sample preparation workflow, we demonstrated deep proteome profiling of >6000 protein groups from small populations of cells, including ∼650 HeLa cells and 10 single human pancreatic islet thin sections (∼1000 cells) from a pre-symptomatic type 1 diabetic donor.
Highlights
Global proteomic analyses, in which the entire complement of proteins expressed in a biological system is identi ed and quanti ed to the extent practicable, continue to support systems-level inquiry and inform on protein changes in response to environmental perturbations, spatial distribution, developmental state, etc.[1,2,3,4] In addition to advancing basic research, in-depth global proteomics can enable extensive functional mapping of proteoforms arising from a speci c gene, holding great potential for personalized and precision medicine.[5]
Sensitivity limitations of mass spectrometry (MS)-based proteomics have led to minimum sample requirements of thousands or millions of cells, which precludes the indepth molecular characterization of limited clinical samples, including ne needle aspiration biopsies, puri ed primary cells and circulating tumor cells
The peptides eluted from the high-pH RPLC separation were concatenated at 1 minute intervals into 4 or 12 nanowells on another micro uidic glass chip using an in-house-developed robotic system (Fig. 1b and Video S1†) and allowed to dry
Summary
In which the entire complement of proteins expressed in a biological system is identi ed and quanti ed to the extent practicable, continue to support systems-level inquiry and inform on protein changes in response to environmental perturbations, spatial distribution, developmental state, etc.[1,2,3,4] In addition to advancing basic research, in-depth global proteomics can enable extensive functional mapping of proteoforms arising from a speci c gene, holding great potential for personalized and precision medicine.[5]. The nanowell platform minimizes peptide losses to surfaces in offline 2D LC fractionation, enabling >5800 proteins to be confidently identified from just 50 ng of HeLa digest.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
