title: High-throughput SISCAPA quantitation of peptides from human plasma digests by ultrafast, liquid chromatography-free mass spectrometry.
abstract: We investigated the utility of an SPE-MS/MS platform in combination with a modified SISCAPA workflow for chromatography-free MRM analysis of proteotypic peptides in digested human plasma. This combination of SISCAPA and SPE-MS/MS technology allows sensitive, MRM-based quantification of peptides from plasma digests with a sample cycle time of ∼7 s, a 300-fold improvement over typical MRM analyses with analysis times of 30-40 min that use liquid chromatography upstream of MS. The optimized system includes capture and enrichment to near purity of target proteotypic peptides using rigorously selected, high affinity, antipeptide monoclonal antibodies and reduction of background peptides using a novel treatment of magnetic bead immunoadsorbents. Using this method, we have successfully quantitated LPS-binding protein and mesothelin (concentrations of ∼5000 ng/mL and ∼10 ng/mL, respectively) in human plasma. The method eliminates the need for upstream liquid-chromatography and can be multiplexed, thus facilitating quantitative analysis of proteins, including biomarkers, in large sample sets. The method is ideal for high-throughput biomarker validation after affinity enrichment and has the potential for applications in clinical laboratories.
authors.affiliation: Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia, Canada.
authors.forename: Morteza
authors.initials: M
authors.lastname: Razavi
authors.forename: Lauren E
authors.initials: LE
authors.lastname: Frick
authors.forename: William A
authors.initials: WA
authors.lastname: LaMarr
authors.forename: Matthew E
authors.initials: ME
authors.lastname: Pope
authors.forename: Christine A
authors.initials: CA
authors.lastname: Miller
authors.forename: N Leigh
authors.initials: NL
authors.lastname: Anderson
authors.forename: Terry W
authors.initials: TW
authors.lastname: Pearson
mesh_terms: D000209:Acute-Phase Proteins / Q000032:analysis; D000911:Antibodies, Monoclonal / Q000737:chemistry; D000915:Antibody Affinity; D015415:Biomarkers / Q000097:blood; D001798:Blood Proteins / Q000032:analysis*; D002352:Carrier Proteins / Q000032:analysis; D002853:Chromatography, Liquid; D058851:GPI-Linked Proteins / Q000097:blood; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008562:Membrane Glycoproteins / Q000032:analysis; D000090204:Mesothelin; D010455:Peptides / Q000097:blood*; D040901:Proteomics / Q000379:methods; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012984:Software; D013997:Time Factors; D000099231:Lipopolysaccharide-Binding Protein
chemical_list: D000209:Acute-Phase Proteins; D000911:Antibodies, Monoclonal; D015415:Biomarkers; D001798:Blood Proteins; D002352:Carrier Proteins; D058851:GPI-Linked Proteins; D008562:Membrane Glycoproteins; D000090204:Mesothelin; D010455:Peptides; D000099231:Lipopolysaccharide-Binding Protein