Application Note 53
Selection and Synthesis to Analysis: Stable Isotope-Labeled Peptides for Proteomic Applications
Andrew J. Percy1, Sandeep Singh2, Travis White2
1. Cambridge Isotope Laboratories, Inc. Tewksbury, MA USA
2. CEM Corporation, Matthews, NC USA
Introduction
Proteomics is among the core ‘omics technologies commonly implemented in the scientific field to study the molecular processes that drive cellular function and disease pathogenesis. While this enabling technology can be employed independently, it is being increasingly combined today with other ‘omics technologies, such as metabolomics, to capture a more comprehensive view of systems biology.1,2 Regardless of the ‘omics technology deployed, mass spectrometry (MS) is the ideal tool for analyte detection (peptides being the focus here) due to its wide-spanning array of analytical benefits. At the sample analysis stage, a mass spectrometer can be operated in a variety of modes (e.g., multiple reaction monitoring, MRM; parallel reaction monitoring, PRM) and is commonly fronted by a separation technique (e.g., liquid chromatography or LC) for enhanced specificity and sensitivity of measurement.
One of the critical elements of method development is the sample preparation. The method used should incorporate isotopically labeled standards (e.g., 13C and/or 15N) as these can help compensate for matrix-induced suppression and procedural/instrumental variation. Additional benefits of these standards are that they assist in correctly identifying endogenous analytes, detecting chemical interference, and improving analytical precision. In a proteomics application, the choice of normalizer (i.e., peptide or protein labeled standard) is user-based, with each carrying its inherent advantages and disadvantages that should be weighed. The stable isotope-labeled peptide standard approach is common in MS proteomics, particularly in targeted biomarker research,3 and has proven through the years to be a reliable means to qualify and quantify peptides (and proteins by inference). To ensure robust assay workflows, certain considerations must be borne in the experimental design and application development. This pertains, for example, to the selection, synthesis, and analysis of the stable isotope-labeled peptide standards.
In this article, two stable isotope-labeled peptide standards from two recent (and dissimilar) publications from the proteomics field were selected. The case examples were peptides ATHVGNDTTLAQIVK (13C6/15N2 at K or L-lysine) and AYNVTQAFGR (13C3 at A or L-alanine and 13C6/15N4 at R or L-arginine). In their applications, the ATHVGNDTTLAQIVK labeled standard was used to quantify the ATP7B protein in dried blood spot samples of Wilson’s disease patients.4 The latter AYNVTQAFGR standard was used in the targeted LC-PRM/MS analysis of clinical nasopharyngeal swab samples of COVID-19 patients.5 Example methods for their synthesis and characterization is described herein along with a summary of the labeled standard application in MS-based proteomics research.
Highlights
• Guidelines and considerations for peptide selection, synthesis, and analysis in bottom-up proteomic applications are described
• Isotopically labeled peptide standards from two published research articles were selected for reference and relevance
• Outlined synthesis procedures utilized CIL’s stable isotope-labeled protected amino acids and were performed on CEM’s Liberty PRIME™ 2.0 peptide synthesizer
• Qualitative and quantitative applications of the spotlighted proteomic articles are summarized
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