The Thermo Scientific Pierce Peptide Retention Time Calibration Mixture enables reversed phase liquid chromatography (LC) and mass spectrometry (LC-MS) users to optimize and confirm correct operation of their equipment.
The Pierce Peptide Retention Time Calibration Mixture is used to optimize and assess LC parameters, identify total peptide elution window and optimize MS parameters. Testing the mixture frequently throughout the lifetime of a column or including it in quantitative samples makes it possible to monitor and normalize LC-MS performance between samples and over time.
When used with Thermo Scientific Pinpoint 1.1 software, the calibration mixture can predict peptide retention times based upon peptide sequence or relative retention time between instruments. The mixture can also be used with Pinpoint software to establish normalized collision energies between platforms. This allows normalization of methods and eases the transfer of quantitative methods between LC-MS platforms. Pinpoint software, sample files, tutorials, and videos can be downloaded from the Thermo Proteomics Portal.
- Assess chromatography and MS instrument performance
- Predict peptide retention across multiple instrument platforms
- Predict peptide retention time from sequence using calculated hydrophobicity factor
- Optimize scheduled MS acquisition windows for improved quantification and increased multiplexing
- Normalize collision energies between instruments and platforms
- Normalize results for variation in retention times and peak intensities between runs
The Pierce Peptide Retention Time Calibration Mixture contains 15 synthetic heavy peptides mixed at an equimolar ratio that elute across the chromatographic gradient. The observed retention times and hydrophobicity factors (HF) for these calibrant peptides are fit to a linear equation to determine the slope of the retention time/HF relationship. This equation and the HF of an uncharacterized peptide are then used to predict retention time.
|Thermo Scientific Pierce Peptide Retention Time Calibration Mixture Components and Properties. The peptide sequences, peptide masses and chromatographic behavior of each component of the Peptide Retention Time Calibration Mixture are given above. The position and identify of the heavy isotope-labeled amino acid in each sequence is indicated in bold.
The prediction of peptide retention time assists in the development of multiplexed, high throughput mass spectrometric assays. The Pierce Peptide Retention Time Calibration Mixture and Thermo Scientific Pinpoint software streamline the transition from qualitative protein discovery results to the development of targeted MS assays on Thermo Scientific triple quadrupole, Orbitrap, Exactive, and ion trap mass spectrometers. The Pierce Peptide Retention Time Calibration Mixture permits optimization and regular assessment of chromatographic performance and for rapid development of multiplexed, scheduled targeted MS assays for the quantification of dozens to hundreds of peptide targets per run.
|Chromatographic Analysis of the Thermo Scientific Pierce Peptide Retention Time Calibration Mixture. A.The Peptide Retention Time Calibration Solution (250fmol) was analyzed in duplicate on a Thermo Scientific LTQ XL Orbitrap Mass Spectrometer using a self-packed column (75µm x 20cm) containing Magic* C18 (Michrom Bioresources) using a 0.25% per minute gradient of Buffer A (0.1% formic acid) and Buffer B (0.1% formic acid/99.9 % acetonitrile) at 300nL per minute. B. The Retention Time Calibration Solution was also analyzed on a Thermo Scientific TSQ Vantage Mass Spectrometer using a Thermo Scientific Hypersil Gold C18 column (1.0 x 150mm, Part No. 25005-150165) with a 1.0% per minute gradient at 120µL per minute. Numbered peaks correspond to the calibrant peptides described above.
FASTA-formatted sequences of Peptide Retention Time Calibration Mixture peptides (download zip file)
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Review of Mass Spectrometry
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