{"id":8608,"date":"2016-05-25T11:00:49","date_gmt":"2016-05-25T11:00:49","guid":{"rendered":"http:\/\/admin.acceleratingscience.com\/proteomics\/?p=8608"},"modified":"2016-05-25T11:00:49","modified_gmt":"2016-05-25T11:00:49","slug":"data-acquisition-revamp-for-large-scale-prm-proteomics","status":"publish","type":"post","link":"https:\/\/www.thermofisher.com\/blog\/proteomics\/data-acquisition-revamp-for-large-scale-prm-proteomics\/","title":{"rendered":"Data Acquisition Revamp for Large-Scale PRM Proteomics"},"content":{"rendered":"

\"SampleGallien<\/span><\/span> et al. (2015) provide a methodologically dense paper that sets out <\/span><\/span>the protocols for internal standard triggered–parallel reaction monitoring (IS-PRM), <\/span><\/span>a novel <\/span><\/span>targeted <\/span><\/span>data acquisition scheme for large-scale <\/span><\/span>proteomics. In essence, the authors<\/span><\/span> present a validated system for <\/span><\/span>real-time instrument <\/span><\/span>operation and <\/span><\/span>data acquisition control<\/span><\/span>,<\/span><\/span> <\/span><\/span>in addition to <\/span><\/span>quantitat<\/span><\/span>ive<\/span><\/span> proteomics analysis for a larger target grouping. <\/span><\/span>Ultimately, the research team posits that <\/span><\/span>IS-PRM<\/span><\/span> presents advantages over both serial reaction monitoring (SRM)<\/span><\/span>, and<\/span><\/span> existing direct data acquisition (DDA) and targeted data acquisition (TDA)<\/span><\/span> modes<\/span><\/span>.<\/span><\/span> <\/span><\/p>\n

Using the drive toward biomarker discovery<\/span><\/span> as an incentive<\/span><\/span> where scientists analyze larger and larger peptide numbers under increasingly efficient instrument o<\/span><\/span>peration<\/span><\/span>, <\/span><\/span>Gallien<\/span><\/span> et al. propose <\/span><\/span>a<\/span><\/span> targeted workflow with<\/span><\/span> internal standard monitoring switching mass spectrometry operation between “watch mode” and “quantitative mode” for data acquisition. The team accomplished this with stable isotope labeled <\/span><\/span>(SIL) <\/span><\/span>internal peptide references spiked into experimental samples. In this way, they describe instrument control as dynamic and occurring in real time through continual monitoring of the tandem <\/span><\/span>mass<\/span><\/span> spectrometry (MS\/MS) spectra generated during each experimental run.<\/span><\/span> <\/span><\/p>\n

The basis of the IS-PRM workflow is the use<\/span> of SIL peptides to generate internal standards that are common across all experimental protein digests. The team employed<\/span> various sources—including <\/span><\/span>PEPotec<\/a> and <\/span><\/span>Pierce Peptide Retention Time Calibration Mix<\/a><\/span><\/span> (both <\/span><\/span>Thermo<\/span><\/span> Scientific)—as internal standards to spike human plasma<\/span><\/span>, urine or pooled HeLa cell<\/span><\/span> peptide digests prior to liquid chromatography (LC)-MS\/MS<\/span><\/span> <\/span><\/span>evaluation. In addition, the researchers also used a series of 13 SIL peptides as external references for instrument calibration between and within run times.<\/span><\/span> <\/span><\/p>\n

Gallien<\/span><\/span> et al. used a number of chromatography systems and quadrupole-O<\/span><\/span>rbitrap<\/span><\/span> mass spectrometers during validation and operational analysis of the IS-PRM workflow. These included an UltiMate 3000 <\/a><\/span><\/span>RSLCnano<\/span><\/span><\/a> LC system<\/a> in conjunction with Acclaim <\/a><\/span><\/span>PepMap<\/span><\/span><\/a> trap and analytical columns (all <\/span><\/span>Thermo<\/span><\/span> Scientific) for chromatographic separation. Following this, they analyzed the separated peptides on three different <\/span><\/span>quadrupole-O<\/span><\/span>rbitrap<\/span><\/span> <\/span><\/span>mass spectrometers—Q <\/a><\/span><\/span>Exactive<\/a>, <\/span><\/span>Q <\/span><\/span>Exactive<\/span><\/span><\/a> Plus<\/a> or Q Exactive HF<\/a><\/span><\/span> mass spectrometers (all Thermo Scientific)—to generate spectral libraries. The team also used a triple quadrupole instrument, the TSQ Vantage mass spectrometer<\/a> (<\/span><\/span>Thermo<\/span><\/span> Scientific) for <\/span><\/span>S<\/span><\/span>RM assay.<\/span><\/span> <\/span><\/p>\n

Once the team analyzed the spectral data with <\/span><\/span>Xcalibur<\/span><\/span><\/a> software v.2.2 <\/a>and Pinpoint software v.1.3<\/a> (both <\/span><\/span>Thermo<\/span><\/span> Scientific), they also developed programming using C# for in-house scripts and an <\/span><\/span>application programming<\/span><\/span> interface (API) to automate data evaluation in real time.<\/span><\/span> <\/span><\/p>\n

Gallien<\/span><\/span> et al. approached the study in four phases:<\/span><\/span> <\/span><\/p>\n