Jizu Yi

Intrinsic peptidase activity causes a sequential multi-step reaction (SMSR) in digestion of human plasma peptides.

Human plasma and serum samples, including protein and peptide biomarkers, are subjected to preanalytical variations and instability caused by intrinsic proteases. In this study, we directly investigated the stability of peptide biomarkers by spiking an isotopically labeled peptide into human plasma and serum samples and then monitoring its time-dependent change. Fibrinogen peptide A (FPA) was used as a model substrate, and its degradation in a conventional serum and plasma either with citrate, heparin, or EDTA as the anticoagulant, or EDTA plus protease inhibitors (inhibited plasma), was measured using time-course MALDI-TOF MS analysis. The FPA and other peptides tested in this study vary in these samples. However, the peptides are most stable in the inhibited plasma followed by, in general order, EDTA plasma, citrate plasma, heparin plasma and serum, demonstrating the benefit of plasma versus serum, and protease inhibitors for biomarker stabilization. Kinetic analysis indicates that intrinsic peptidases cause an observed first-order Sequential Multiple-Step Reaction (SMSR) in digestion of the peptide. Modeling analysis of the SMSR demonstrates that step reactions differ in their kinetic rate constants, suggesting a significant contribution of the truncated end residue on the substrate specificity of the intrinsic peptidase(s). Our observations further show that synthetic peptides introduced into plasma as internal controls can also be degraded, and thus, their (in)stability as a preanalytical variable should not be overlooked.

Thrombin induces broad spectrum proteolysis in human serum samples.

Abstract Background: During clotting, α thrombin cleaves fibrinogen releasing fibrinopeptide A (FPA). FPA is easily identified in serum using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). Using MALDI-TOF MS, we observed multiple, progressively shorter fragments of serum FPA. Following ambient incubation of serum, variations in the content of FPA fragments occur over time. Denaturation of α thrombin by heating the serum sample appears to minimize this variation. These observations suggest that intrinsic proteolytic and peptidolytic activity is elevated in serum and perhaps originates from the coagulation cascade enzymes themselves, especially α thrombin. Methods: Extrinsic addition of α thrombin to a subset (3–30 kDa) of plasma proteins was carried out to induce proteolysis and to examine the resultant peptides to reveal α thrombin susceptible parent proteins. One of these identified proteins, hemopexin, was directly digested by α thrombin and the peptides examined to confirm the observations from the initial plasma protein digestion. Results: Extrinsic addition of α thrombin to a subset (3–30 kDa) of plasma proteins results in wide-spread digestion of proteins unrelated to coagulation, revealing a substrate range encompassing more than fibrinogen. Direct digestion of one of these proteins, hemopexin, by α thrombin confirms these observations. Conclusions: The resulting peptides indicate broad tolerance beyond the consensus R-G cleavage site of fibrinogen; in fact, there appears to be no bias for the amino acid following the R/K residue. These data support our hypothesis that the enzymatic activities inherent to coagulation, or at least to thrombin, contribute to destabilization of the protein and peptide content of serum. Clin Chem Lab Med 2009;47:685–93.

Minimizing Preanalytical Variation of Plasma Samples by Proper Blood Collection and Handling

Blood samples collected for proteome studies are subject to a variety of preanalytical instability, among which intrinsic proteolysis activities cause a broad spectrum of protein and peptide degradation. This chapter describes two MALDI MS-based methods for plasma peptidomic analyses; a direct MALDI-TOF MS and an LC MALDI-TOF MS. Using these methods, we compared peptides and their time-dependent changes in traditional serum, four plasma samples with different anticoagulants and additives: EDTA-based, citrate-based, or heparin-based, and EDTA-based with protease inhibitors. For minimizing plasma sample instability and preanalytical variation, we suggest using an optimized blood collection device, minimizing the dwell time during blood collection and handling, controlling centrifugation and handling at room temperature, and saving plasma samples for use at most one freeze/thaw cycle. We have optimized our protocol to achieve reproducibility in peptidomic analyses of plasma samples using MALDI-TOF MS by minimizing preanalytical and analytical variability.

Degradation and Stabilization of Peptide Hormones in Human Blood Specimens.

Plasma hormone peptides, including GLP-1, GIP, Glucagon, and OXM, possess multiple physiological roles and potential therapeutic and diagnostic utility as biomarkers in the research of metabolic disorders. These peptides are subject to proteolytic degradation causing preanalytical variations. Stabilization for accurate quantitation of these active peptides in ex vivo blood specimens is essential for drug and biomarker development. We investigated the protease-driven instability of these peptides in conventional serum, plasma, anticoagulated whole blood, as well as whole blood and plasma stabilized with protease inhibitors. The peptide was monitored by both time-course Matrix-Assisted Laser Desorption Ionization Time-to-Flight Mass Spectrometry (MALDI –TOF MS) and Ab-based assay (ELISA or RIA). MS enabled the identification of proteolytic fragments. In non-stabilized blood samples, the results clearly indicated that dipeptidyl peptidase-IV (DPP-IV) removed the N-terminal two amino acid residues from GLP-1, GIP and OXM(1-37) and not-yet identified peptidase(s) cleave(s) the full-length OXM(1-37) and its fragments. DPP-IV also continued to remove two additional N-terminal residues of processed OXM(3–37) to yield OXM(5–37). Importantly, both DPP-IV and other peptidase(s) activities were inhibited efficiently by the protease inhibitors included in the BD P800* tube. There was preservation of GLP-1, GIP, OXM and glucagon in the P800 plasma samples with half-lives > 96, 96, 72, and 45 hours at room temperature (RT), respectively. In the BD P700* plasma samples, the stabilization of GLP-1 was also achieved with half-life > 96 hours at RT. The stabilization of these variable peptides increased their utility in drug and/or biomarker development. While stability results of GLP-1 obtained with Ab-based assay were consistent with those obtained by MS analysis, the Ab-based results of GIP, Glucagon, and OXM did not reflect the time-dependent degradations revealed by MS analysis. Therefore, we recommended characterizing the degradation of the peptide using the MS-based method when investigating the stability of a specific peptide.

Investigation of peptide biomarker stability in plasma samples using time-course MS analysis.

Peptide biomarkers in plasma or serum are subject to proteolytic degradation caused by intrinsic peptidase activities, resulting in a potential barrier in translating a discovered biomarker into clinical application. This chapter describes a method using time-course MALDI-TOF MS analysis to investigate the stability of a plasma peptide biomarker under a variety of preanalytical situations. A synthesized peptide with the same primary sequence as a potential endogenous biomarker is spiked into a blood sample, and the sample is incubated over time at r.t. (25  ±  1°C) or other preanalytical situations. At a specific period of incubation time, the sample is quenched with the addition of acid with or without an internal control peptide. The spiked peptides in the sample are extracted with one of three procedures for highly soluble, moderately soluble, or essentially insoluble peptides. The peptide samples are then analyzed using MALDI-TOF MS. The abundance changes of the peptide biomarker are monitored by time-course changes of the mass spectra. These changes over-time are measured and fitted to a first-order degradation reaction so that stability of the peptide biomarker (half-life) can be calculated. Kinetics analysis of both parent and shorter (daughter) peptides are also possible by fitting to a sequential multiple-step reaction (SMSR) model. This optimized method facilitates evaluation of biomarker stability, and helps to define sample handling and analytical processing steps that contribute to instability of measured peptide biomarker(s).

Time-Dependent and Sample-to-Sample Variations in Human Plasma Peptidome are Both Minimized Through Use of Protease Inhibitors

Abstract In the current study, we have developed a method to measure relative peptide stability over time in different blood collection tubes. Reversed-phase chromatography and liquid chromatography–matrix-assisted laser desorption/ionization were performed on three subjects to facilitate a deeper look into the plasma peptidome. The data further support the importance of protease inhibitors in stabilizing plasma samples. Significantly, we have revealed subject-to-subject variability in the intrinsic damage over time that is possible in standard EDTA plasma, with such variations extensively minimized by protease inhibitors. We conclude that protease inhibitors can simultaneously improve time-dependent and individual-dependent preanalytical variables in human plasma samples.

Abstract 365: Stabilization of incretin in human plasma sample

Incretin peptides, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), possess multiple physiological roles that make them important peptide biomarkers for metabolic disorder. GLP-1 and GIP contribute to approximately 60 -70 % of the total postprandial insulin response in healthy individuals, and potentially have a therapeutic value in treatment of type II diabetes. Glucagon-like peptides (GLP-1, and GLP-2) regulate cell proliferation, differentiation, and apoptosis. GLP-1 also contributes to physiological roles in controlling energy homoestasis and balance through both peripheral signals and brain stem regulations of appetite in the nucleus of the solitary tract (NTS). Circulating GLP-1 acts to inhibit food intake, acute GLP-1 reduces calorie intake, and GLP-1 neuronal circuits of NTS may have anorectic effect in energy homeostasis. However, the incretin effect is significantly decreased in patients with type 2 diabetes, delaying and reducing insulin release after oral glucose administration. It is well documented that circulating GLP-1 and GIP after their secretion are rapidly digested by the intrinsic blood enzyme dipeptidyl peptidase-IV (DPP-IV). The half-life of these two peptides in in vivo plasma is very short: approximately 2 minutes for intact GLP-1 and 5 minutes for intact GIP. Ex vivo stabilization of full functions of these peptides is critical for their utility in diagnostics and/or drug development. We directly investigated the stability of these peptides and their metabolites in conventional serum, EDTA plasma, or EDTA plasma with protease inhibitors as stabilizers. Using spiked blood samples, peptides were monitored by time-course MALDI-TOF MS. Mass spectrometry allows detection of the intact peptides for kinetic analysis as well as identification of the cleaved peptides. The results indicate that degradation of GLP-1 fits to a simple Michaelis-Menton Equation (K m = 21.36 nM and V m = 7.05 nM), while GIP fits an observed first-order reaction, and both having the greatest stability in the inhibited plasma sample. The half-life of GLP-1 peptide was also dependence of the peptide concentration, with lower concentration having less half-life time. Identification of the cleaved peptides indicates that both intrinsic DPP-IV and exo-carboxypeptidase contribute to the digestion of active GLP-1 in ex vivo sample. Further, our study indicates that stabilization of both GLP-1 and GIP was accomplished by including a cocktail of enzyme inhibitors in a blood-collection tube, allowing accurate measurement for biomarker development. We also conducted an evaluation of two commercially available ELISA kits for GLP-1 and GIP demonstrating some kits are sensitive to peptidase degradation while others are not. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 365. doi:10.1158/1538-7445.AM2011-365