Tao Liu — Page 10 | Search PubMed

PubMedDate not supplied

Utilizing human blood plasma for proteomic biomarker discovery.

Candidate proteomic biomarker discovery from human plasma holds both incredible clinical potential as well as significant challenges. The dynamic range of proteins within plasma is known to exceed 10(10), and many potential biomarkers are likely present at lower protein abundances. At present, proteomic based MS analyses provide a dynamic range typically not exceeding approximately 10(3) in a single spectrum, and approximately 10(4)-10(6) when combined with on-line separations (e.g., reversed-phase gradient liquid chromatography), and thus are generally insufficient for low level biomarker detection directly from human plasma. This limitation is providing an impetus for the development of experimental methodologies and strategies to increase the possible number of detections within this biofluid. Discussed is the diversity of available approaches currently used by our laboratory and others to utilize human plasma as a viable medium for biomarker discovery. Various separation, depletion, enrichment, and quantitative efforts as well as recent improvements in MS capabilities have resulted in measurable improvements in the detection and identification of lower abundance proteins (by approximately 10-10(2)). Despite these improvements, further advances are needed to provide a basis for discovery of candidate biomarkers at very low levels. Continued development of depletion and enrichment techniques, coupled with improved pre-MS separations (both at the protein and peptide level) holds promise in extending the dynamic range of proteomic analysis.

Biomarkers↗

PubMedDate not supplied

Human plasma N-glycoproteome analysis by immunoaffinity subtraction, hydrazide chemistry, and mass spectrometry.

The enormous complexity, wide dynamic range of relative protein abundances of interest (over 10 orders of magnitude), and tremendous heterogeneity (due to post-translational modifications, such as glycosylation) of the human blood plasma proteome severely challenge the capabilities of existing analytical methodologies. Here, we describe an approach for broad analysis of human plasma N-glycoproteins using a combination of immunoaffinity subtraction and glycoprotein capture to reduce both the protein concentration range and the overall sample complexity. Six high-abundance plasma proteins were simultaneously removed using a pre-packed, immobilized antibody column. N-linked glycoproteins were then captured from the depleted plasma using hydrazide resin and enzymatically digested, and the bound N-linked glycopeptides were released using peptide-N-glycosidase F (PNGase F). Following strong cation exchange (SCX) fractionation, the deglycosylated peptides were analyzed by reversed-phase capillary liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Using stringent criteria, a total of 2053 different N-glycopeptides were confidently identified, covering 303 nonredundant N-glycoproteins. This enrichment strategy significantly improved detection and enabled identification of a number of low-abundance proteins, exemplified by interleukin-1 receptor antagonist protein (approximately 200 pg/mL), cathepsin L (approximately 1 ng/mL), and transforming growth factor beta 1 (approximately 2 ng/mL). A total of 639 N-glycosylation sites were identified, and the overall high accuracy of these glycosylation site assignments as assessed by accurate mass measurement using high-resolution liquid chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR) is initially demonstrated.

Amino Acid Motifs↗