Application Note 50
Translation and Implementation of the PeptiQuant™ Plus Human Plasma BAK-270
Andrew J. Percy,1 Ryan Trouvé,2 Sylvain Lehmann,2 Christophe Hirtz,2 Jérôme Vialaret2
1. Cambridge Isotope Laboratories, Inc., Tewksbury, MA USA
2. Université de Montpellier, CHU de Montpellier, Laboratoire de Biochimie Protéomique Clinique, Montpellier, France
Abstract
The large-scale quantitation of proteins in biomarker screening exercises is being increasingly employed in the proteomics field to discover, and ultimately validate, disease indicators. These evaluations are commonly performed in blood plasma samples by liquid chromatography (LC)-tandem mass spectrometry (MS/MS). To assist researchers developments and applications, Cambridge Isotope Laboratories, Inc. (CIL) supplies PeptiQuant™ Plus biomarker assessment kits (BAKs) for plasma protein interrogations by bottom-up LC-MS/MS. As part of a joint collaboration with CIL and Université de Montpellier, the latest BAK-270 was translated to a Shimadzu LC-MS platform at the Université de Montpellier. This application note outlines that process, its results, and an overview into its clinical application at Montpellier Hospital.
Keywords
Human plasma, human serum, biomarkers, multiplex, LC-MRM
Introduction
Considerable efforts are being leveraged year over year to discover and validate protein biomarkers using MS-based quantitative approaches. The motivation for this lies in personalized or precision medicine whereby biomarkers (i.e., biological indicators) are sought for enhanced disease monitoring, companion diagnostics, and improved patient outcomes. Quantitation can be accomplished in a number of ways, with the use of targeted MS/MS (acquisition mode: multiple reaction monitoring, MRM) and stable isotope-labeled standards (SIS, be it peptides or proteins) being a popular implementation within a bottom-up proteomics regime. In its most basic orientation, an MRM with SIS peptide approach can quantify multiplexed panels of plasma proteins, many of which have putative or approved association(s) to disease and cancer. In this type of method operation, the labeled standards are constructed to resemble the chemical structure of their endogenous (natural or NAT) analogue.
MS screening studies can be aided by commercialized kits that have been validated for their intended use. One type of kit is PeptiQuant Plus, with the biomarker assessment line designed for the precise and rapid quantitation of proteins (e.g., 270 using 270 pep tides as surrogates) in undepleted and nonenriched human plasma.1,2 Favorable to the PeptiQuant kits is that they have been rigorously characterized according to the complete set of CPTAC (Clinical Proteomic Tumor Analysis Consortium) guidelines.3 To help facilitate standardization, the kits are supplied with key materials and tools for broad implementation. While the kits are already optimized to select LC-MS platforms, this may not be applicable to all users’ experimental setups.
In this note, the procedure and test results for translating an off-the-shelf PeptiQuant Plus BAK-270* to the clinical proteomics laboratory at Montpellier Hospital is described. The full co-author list has experience in this type of translation having successfully adapted previous PeptiQuant kits to other platforms for alternate quantitative proteomic sample applications.4-8 The supplied materials in the BAK-270 were all utilized in the development and translation, with the final bottom-up LC-MRM/MS method ultimately applied to human plasma/serum samples. Note that while the defined translation here is for a Shimadzu platform (comprises Shim-pack GISS-HP HPLC column coupled to an 8060 triple quadrupole mass spectrometer), other LC-MS arrangements should be extendable.
Read more by downloading the application note.
*PeptiQuant is a trademark of MRM Proteomics Inc., the commercial product originator.
