Targeted Protein Quantitation - The next step in the search for biomarkers A common endpoint for a biomarker discovery experiment is a list of putative marker proteins. A common next step is to target these proteins for quantitative measurement in an expanded patient population to verify their statistical significance. Unlike in the discovery phase, assays with hundreds of samples need to be run for statistical verification. A robust, high-throughput quantitative assay that offers both specificity and sensitivity is required. Selected reaction monitoring for targeted quantitation The most common analytical approach to targeted protein quantitation is to use selected-reaction monitoring (SRM) on a triple quadrupole mass spectrometer to monitor a unique peptide or group of unique peptides for each protein. SRM on a triple quad provides an outstanding combination of specificity, sensitivity, and reliability, making it ideal for this application. Tandem mass tags that enable concurrent identification and quantitation of proteins in different samples are sometimes incorporated into this workflow. Intelligent SRM facilitates assay development Developing an SRM assay for targeted proteins can be challenging. One of the main obstacles is finding peptides that are sequence specific or unique to a targeted protein and that also give sufficient signal intensity. If the list of targeted proteins is long, the initial list of candidate peptides can be extensive. The list of SRM (MS/MS) transitions, which typically includes several transitions for each peptide, can easily exceed one thousand. To quickly find the best prototypic peptides and the best SRM transition(s) for each peptide, a triple quadrupole system must be able to survey hundreds of peptides, with proof of their identities, in a single HPLC-MS run. To address this challenge, intelligent SRM (iSRM) technology has been incorporated into the Thermo Scientific TSQ family of triple quadrupole mass spectrometers. iSRM uses a combination of time-based and data-dependent SRM to optimize the use of precious acquisition time and increase productivity without significantly compromising data quality. iSRM technology enables processing of over 15,000 SRM transitions for simultaneous quantification and identity confirmation of up to 1000 peptides in a single HPLC-MS run. It dramatically reduces the labor and cost of developing multiplexed SRM assays. Pinpoint software for a seamless transition from discovery to targeted quantitation Thermo Scientific Pinpoint was created specifically to simplify the creation of iSRM assays. Using data from discovery experiments, PinpointTM software largely automates the preliminary selection of SRM transitions to be monitored. It predicts proteotypic peptides, determines the best SRM transitions, and builds the TSQ instrument method. The iSRM workflow development process is iterative. The preliminary method is used to acquire data, which is in turn used to refine the method. Pinpoint software also provides tools for evaluating this preliminary data. The target peptides that show the best responses are retained while target peptides that do not show good responses are removed from the final iSRM method. This ensures that the limited, fixed cycle time is used to analyze only peptides that provide high-quality results. View a recorded webinar describing the interaction of iSRM and Pinpoint software Obtain more information and download Pinpoint software here. The high-resolution advantage Although SRM by triple quadrupole MS gives very specific and sensitive responses for targeted peptides, the resolution limit of the first quadrupole (Q1) can make it difficult to differentiate between a targeted precursor and chemical background or a non-targeted peptide, particularly when detecting very-low-abundance peptides in highly complex samples. It can also prevent the use of valuable precursor-to-product transitions if other precursor peptides in the sample have similar masses and product ions. The Thermo Scientific TSQ Quantum Ultra and TSQ Vantage triple quadrupole mass spectrometers provide an effective solution to this challenge. Whereas most triple quads are limited to unit mass resolution (0.7 Da FWHM) for precursor selection, the TSQ Quantum UltraTM and TSQ VantageTM can employ high-resolution precursor selection (0.2 Da FWHM). The additional specificity of this high-resolution selected reaction monitoring (H-SRM) reduces interferences, providing more accurate quantitation and more robust assays without loss of signal. Compared to competing instruments, it reduces the need to discard transitions that would otherwise provide additional specificity. | 
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