Gul Mustafa (Presenter)
Protea Biosciences, Inc.
Authorship: Gul M Mustafa (1), Mark Szewc (1), Ryan Faucette (2 ) and Dan Sexton (2)
(1) Protea Biosciences, 1311 pineview drive Morgantown WV, (2 ) Dyax Corporation, Burlington, MA
| Short Abstract The purpose of this study was to develop a robust LC-MS based peptide quantitation assay to differentiate kininogen (HK) and kallikrein cleaved kininogen in order to determine a 2HK cut-point in plasma of healthy volunteers from patients with disease. MRM on an ABSciex 5500 QTrap mass spectrometer was done on various types of plasma samples. The target candidate peptides (SSRIGE, SSRIGEIKE mis 2, KKIYPTVNC QPLGMISLMKRPPGFSPFRSSRIGE, KKIYPTVNCQPLGMISLMK, and SYYFDLTDGLS) and 3-5 product ions for each peptide were validated empirically for plasma kininogen. Ratio of AUC for each peptide between different sample types was calculated. . The ratio of SSRIGE peptide against HMWK vs LMWK unique peptide SYYFDLTDGLS has a potential to be used in an assay to differentiate plasma of healthy volunteers from patients with disease. |
Long Abstract
Introduction: High molecular weight kininogen (HMWK or HK) is a circulating plasma protein and component of the contact system, the activation of which leads to intrinsic pathway-mediated coagulation as well as generation of the potent vasodilator bradykinin via the Kallikrein-kinin system. HMWK adheres to cell surface receptors on the endothelium monocytes, and platelets thereby localizing coagulation and bradykinin generation. The active peptide bradykinin that is released from HMWK shows a variety of physiological effects, such as smooth muscle contraction, hypotension, diuresis, decrease in blood glucose level, it is a mediator of inflammation and has a cardio protective effect, directly via bradykinin action, indirectly via endothelium-derived relaxing factor action.
The purpose of this study was to assess the technical feasibility to develop a robust LC-MS based peptide quantitation assay to differentiate kininogen (HK) and kallikrein cleaved kininogen (2HK heavy chain and 2HK light chain). Single chain HMWK [HK] and two chain HMWK [2HK] was sourced from Enzyme Research Laboratories for use as standards in method development. Different types of plasma samples (Citrate, Citrate + Protease inhibitor (PI), normal SCAT [sample collection/Anticoagulant tubes] plasma, and HAE SCAT plasma) were used for method development. Quality controls were run between runs and reproducibility checks were performed for processing variability on different days and on different runs.
The overall goal to develop this semi-quantitative assay is to measure the ratio of 2HK product to HK substrate in order to determine a 2HK cut-point in plasma of healthy volunteers from patients with disease.
Methodology: Multiple reaction monitoring (MRM) enables quantification of proteins in complex mixture providing a sensitive and selective tool to validate candidate biomarkers in a disease process. For this approach an ABSciex 5500 QTrap mass spectrometer was used and differences between various types of plasma samples was assessed after method optimization. Individual plasma samples were digested with Glu C and MRM analysis was done on the peptides of interest. Glu C was enzyme of choice to obtain unique peptides differentiating HK from 2HK. The sample types used for MRM analysis were normal and HAE plasma collected in the presence of protease inhibitors (referred to as SCAT plasma), Kininogen deficient plasma, normal citrated plasma, normal citrated plasma activated with factor XIIa and HK standards. Initial experiments were designed to verify quality of the protein standards using multiple reaction monitoring mode for specific peptides. 10µl of sample containing 10µg of protein was loaded onto the C18 column. Precursor and product ions predicted by Skyline that were unique to the Glu C digested plasma kininogen were selected. The target candidate peptides (SSRIGE, SSRIGEIKE mis 2, KKIYPTVNC QPLGMISLMKRPPGFSPFRSSRIGE, KKIYPTVNCQPLGMISLMK, and SYYFDLTDGLS) and 3-5 product ions for each peptide were validated empirically for plasma kininogen. The fragmentation energy, and collision energy were individually optimized to the peptides after using skyline for guidance to perform the MRM. Data was analyzed using Analyst Software (ABSciex). The method development focused on optimizing sample preparation, enzyme digestion on different types of plasma samples and HK standards, chromatography, and mass spectrometry parameters.
Results: Ratio of AUC for each peptide between different sample types was calculated. Due to the confirmation of all peptides including HMWK vs LMWK unique peptide (SYYFDLTDGLS) by MRM on Glu C digested HK standard and samples we have high confidence that the targeted peptides found in plasma are attributed to HK.
A significant increase in SSRIGE peptide in HAE SCAT plasma was observed when compared with normal SCAT plasma and an increase was also observed between plasma activated by FXIIa versus non-activated plasma, with no change in HK peptide; which could be due to presence of LMWK in plasma. Also the ratios between SSRIGE/SYYFDLTDGLS was found to be higher in HAE SCAT plasma samples when compared to normal SCAT plasma samples and also in activated versus non-activated plasma. The relative abundance ratios using ratio for SSRIGE and SYYFDLTDGLS was 4.4 and 8.9 for normal SCAT plasma versus HAE SCAT plasma respectively and 19.02 and 48.63 for Normal citrated and citrate activated by FXIIa respectively.
The kininogen deficient plasma (a negative control) sample showed low intensity peak near the detection limits for SSRIGE peptide which was expected possibly due to presence of LMWK.
We also measure HK long peptide, which contains bradykinin (KKIYPTVNC QPLGMISLMKRPPGFSPFRSSRIGE) in plasma samples using targeted assay (MRM); there was almost no change in HK long peptide between normal SCAT and HAE SCAT plasma samples and between activated and non-activated samples; possibly due to presence of LMWK in plasma.
Conclusion: The ratio of SSRIGE peptide against HMWK vs LMWK unique peptide SYYFDLTDGLS has a potential to be used in an assay to differentiate plasma of healthy volunteers from patients with disease. We have run a small set of samples to obtain the cutoff between normal plasma versus HAE plasma. We are in a process of running a large cohort of samples to verify the assay.
References & Acknowledgements:
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