Extending the Depth of Human Plasma Proteome Coverage Using Simple Fractionation Techniques

Abstract

Human plasma is one of the most widely used tissues in clinical analysis, and plasma-based biomarkers are used for monitoring patient health status and/or response to medical treatment to avoid unnecessary invasive biopsy. Data-driven plasma proteomics has suffered from a lack of throughput and detection sensitivity, largely due to the complexity of the plasma proteome and in particular the enormous quantitative dynamic range, estimated to be between 9 and 13 orders of magnitude between the lowest and the highest abundance protein. A major challenge is to identify workflows that can achieve depth of plasma proteome coverage while minimizing the complexity of the sample workup and maximizing the sample throughput. In this study, we have performed intensive depletion of high-abundant plasma proteins or enrichment of lowabundant proteins using the Agilent multiple affinity removal liquid chromatography (LC) columnHuman 6 (Hu6), the Agilent multiple affinity removal LC columnHuman 14 (Hu14), and ProteoMiner followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE) and C18 prefractionation techniques. We compared the performance of each of these fractionation approaches to identify the method that satisfies requirements for analysis of clinical samples and to include good plasma proteome coverage in combination with reasonable sample output. In this study, we report that one-dimensional (1D) gel-based prefractionation allows parallel sample processing and no loss of proteome coverage, compared with serial chromatographic separation, and significantly accelerates analysis time, particularly important for large clinical projects. Furthermore, we show that a variety of methodologies can achieve similarly high plasma proteome coverage, allowing flexibility in method selection based on project-specific needs. These considerations are important in the effort to accelerate plasma proteomics research so as to provide efficient, reliable, and accurate diagnoses, population-based health screening, clinical research studies, and other clinical work.

Deep proteome analysis of plasma reveals novel biomarkers of mild cognitive impairment and Alzheimer’s disease: A longitudinal study

“…Samples were then briefly heated (10 minutes, 70°C), followed by electrophoresis; 1D SDS/PAGE using Invitrogen NuPAGE 4-12% gradient Bis-Tris midi gels (ThermoFisher Scientific, Massachusetts, USA) and 1x Invitrogen MES running buffer according to the manufacturer’s instructions (ThermoFisher Scientific, Massachusetts, USA, USA) followed by colloidal coomassie G250 staining 25 (Figure S4). After destaining, the separated protein lanes were cut evenly with a 24-lane blade (Gel Company Inc., CA), and the gel slices were collected into ten vials for destaining, in-gel trypsin digestion and label-free LCMSMS, following the approach taken in our previously published work 20 .…”

“…The protocol followed for plasma high abundance protein removal, and fractionation of the low abundance proteins, was adapted from a previously published approach 20 . The approach involved depletion of the top 14 high abundance proteins (albumin, IgG, antitrypsin, IgA, transferrin, haptoglobin, fibrinogen, α-2-macroglobulin, α-1-acid glycoprotein, IgM, apolipoprotein AI, apolipoprotein AII, complement C3, and transthyretin) using an Hu14 column (4.6 x 100 mm, Agilent California, United States), followed by SDS/PAGE fractionation of the low abundance protein fraction.…”

Proteomics, Personalized Medicine and Cancer

“…Multidimensional purification has been found to be particularly efficacious, giving high purification factors and reducing sample complexity prior to MS analysis, enabling deeper mining of the proteome [ 13 , 21 , 22 , 23 , 24 ]. As exemplars, Kaur et al have designed a simple fractionation workflow to extend the coverage of the plasma proteome [ 25 ]. In a similar approach, Ahn et al [ 26 ] used a combination of high abundance protein ultradepletion (Agilent MARS-14) and an in-house IgY depletion column, multidimensional peptide fractionation (SCX, SAX, high pH and SEC) and sequential window acquisition of all theoretical mass spectra (SWATH-MS) to screen and identify biomarkers that showed expression alterations in colorectal cancer (CRC) tissues to healthy controls.…”

Proteomics in the pharmaceutical and biotechnology industry: a look to the next decade

“…All of the methods tested performed well, identifying between 3400-3800 plasma proteins. They concluded that the 1D gel-based approach, which allowed for parallel sample processing represented the best choice for high coverage and throughput [156]. Another orthogonal strategy to increase proteome coverage is to utilize enrichment approaches for PTMs.…”