title: Precision of heavy-light peptide ratios measured by maldi-tof mass spectrometry.
abstract: We have investigated the precision of peptide quantitation by MALDI-TOF mass spectrometry (MS) using six pairs of proteotypic peptides (light) and same-sequence stable isotope labeled synthetic internal standards (heavy). These were combined in two types of dilution curves spanning 100-fold and 2000-fold ratios. Coefficients of variation (CV; standard deviation divided by mean value) were examined across replicate MALDI spots using a reflector acquisition method requiring 100 000 counts for the most intense peak in each summed spectrum. The CV of light/heavy peptide centroid peak area ratios determined on four replicate spots per sample, averaged across 11 points of a 100-fold dilution curve and over all six peptides, was 2.2% (ranging from 1.5 to 3.7% among peptides) at 55 fmol total (light + heavy) of each peptide applied per spot, and 2.5% at 11 fmol applied. The average CV of measurements at near-equivalence (light = heavy, the center of the dilution curve) for the six peptides was 1.0%, about 17-fold lower CV than that observed when five peptides were ratioed to a sixth peptide (i.e., a different-sequence internal standard). Response curves across the 100-fold range were not completely linear but could be closely modeled by a power law fit giving R(2) values >0.998 for all peptides. The MALDI-TOF MS method was used to determine the endogenous level of a proteotypic peptide (EDQYHYLLDR) of human protein C inhibitor (PCI) in a plasma digest after enrichment by capture on a high affinity antipeptide antibody, a technique called stable isotope standards and capture by anti-peptide antibodies (SISCAPA). The level of PCI was determined to be 770 ng/mL with a replicate measurement CV of 1.5% and a >14 000-fold target enrichment via SISCAPA-MALDI-TOF. These results indicate that MALDI-TOF technology can provide precise quantitation of high-to-medium abundance peptide biomarkers over a 100-fold dynamic range when ratioed to same-sequence labeled internal standards and enriched to near purity by specific antibody capture. The robustness and throughput of MALDI-TOF in comparison to conventional nano-LC-MS technology could enable currently impractical large-scale verification studies of protein biomarkers.
authors.affiliation: Plasma Proteome Institute , Washington, DC, United States. leighanderson@plasmaproteome.org
authors.forename: N Leigh
authors.initials: NL
authors.lastname: Anderson
authors.forename: Morteza
authors.initials: M
authors.lastname: Razavi
authors.forename: Terry W
authors.initials: TW
authors.lastname: Pearson
authors.forename: Gary
authors.initials: G
authors.lastname: Kruppa
authors.forename: Rainer
authors.initials: R
authors.lastname: Paape
authors.forename: Detlef
authors.initials: D
authors.lastname: Suckau
mesh_terms: D000595:Amino Acid Sequence; D002138:Calibration; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000097:blood / Q000737:chemistry*; D018046:Protein C Inhibitor / Q000097:blood / Q000737:chemistry; D059748:Proteolysis; D040901:Proteomics; D012015:Reference Standards; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods / Q000592:standards; D014357:Trypsin / Q000737:chemistry
chemical_list: D010446:Peptide Fragments; D018046:Protein C Inhibitor; C534934:SERPINA5 protein, human; D014357:Trypsin