Optimize Liquid Chromatography with a Peptide Retention Time Calibration Mixture
Our new mixture of 15 heavy peptides streamlines assay design for targeted peptide quantification
by John C. Rogers, Ph.D.; Michael M. Rosenblatt, Ph.D.; Eugene J. Cichon, B.S.; Scott Peterman, Ph.D.1; Rosa Viner, Ph.D.*; Monica Noonan, M.S. - 06/01/11
The Thermo Scientific Pierce Peptide Retention Time Calibration Mixture enables retention time prediction for peptides on reversed-phase columns. This calibration mixture is useful for predicting peptide retention within a complex mixture for mass spectrometry (MS) analysis.1-3 By testing the mixture throughout the lifetime of a column, it is possible to monitor performance characteristics during column aging. The mixture is also effective for evaluating different reversed-phase supports. column formats, gradients and flow rates.This calibration mixture is also effective for normalizing autosampler variability and chromatographic retention time shifts.
This calibration mixture contains 15 synthetic heavy isotope-labeled peptides mixed at an equimolar ratio that elute throughout the chromatographic gradient. This mixture can be used for optimization and regular assessment of chromatographic performance and for rapid development of multiplexed, scheduled targeted-MS assays for quantifying dozens to hundreds of peptide targets per run.4,5
Assess chromatographic performance of multiple LC platforms
To compare the chromatographic performance of different instrument configurations, the Pierce Peptide Retention Time Calibration Mixture was analyzed on the Thermo Scientific Orbitrap XL and TSQ Vantage Mass Spectrometers configured for discovery or targeted quantitation, respectively. Despite different stationary phases, column formats, gradient conditions and flow rates, both platforms demonstrated reproducible high-resolution peptide separation of the calibration mixture (Figure 1). The elution of peptides throughout the gradient also enabled optimization of chromatographic conditions and performance assessment for multiple runs.
Predict peptide retention time
Peptide retention time depends on peptide hydrophobicity and properties of the liquid chromatography (LC) system. The peptides in the calibration mixture have a wide distribution of hydrophobicity and are effective for calculating the linear relationship between hydrophobicity and observed retention time on any reversed-phase system (Table 1). The Thermo Scientific Pinpoint Software automatically uses the experimentally observed retention times and calculated hydrophobicity of the calibrant to predict retention times for uncharacterized peptides from protein targets.1,2 This approach was used to predict the retention times of 29 peptides from an enzymatic digestion of bovine serum albumin with high accuracy (R2=0.89, Figure 2). Experimentally measured retention times were within 2 minutes of the predicted retention times. This approach uses protein sequence alone or public databases (e.g., Peptide Atlas, www.peptideatlas.org) in the software to develop scheduled, multiplexed MS assays that optimize instrument duty cycle and sensitivity.4,5
Table 1. Thermo Scientific Pierce Peptide Retention Time Calibration Mixture components and properties.
The heavy isotope-labeled amino acid in each sequence is indicated in bold.
|#||Sequence||[M +2H]2+||Hydrophobicity Factor (HF)|
Rapidly develop targeted-MS assays for multiple platforms
Protein MS data collected on a discovery MS platform (e.g., ion trap or Orbitrap Instrument) can be used to create targeted quantitation methods on high-throughput platforms (i.e., triple quadrupole MS instruments). This assay development is streamlined with the help of the calibration mixture. The retention profile of the calibrant on both MS platforms is used by Pinpoint Software to predict relative retention times between platforms. This strategy was used to rapidly develop targeted quantitative MS assays for 35 proteins (478 peptides) identified in a treated cell lysate (Figure 3). The relative retention time method also eases the building of targeted-MS methods for modified peptides, such as phosphopeptides (data not shown).
The Pierce Peptide Retention Time Calibration Mixture is a powerful tool for assessing chromatographic conditions. The retention of uncharacterized peptides can be accurately predicted based on sequence alone. Furthermore, data can be migrated between instruments using relative retention times to ease scheduled MS method development for analyzing dozens to hundreds of peptide targets. The calibration mixture and Pinpoint Software are essential for LC optimization assessment and the development of multiplexed quantitative protein assays.
- Krokhin, O.V. (2006). Sequence-specific retention calculator. Algorithm for peptide retention prediction in ion-pair RP-HPLC: application to 300- and 100-A pore size C18 sorbents. Anal Chem 78(22):7785-95.
- Krokhin, O.V. and Spicer, V. (2009). Peptide retention standards and hydrophobicity indexes in reversed-phase high-performance liquid chromatography of peptides. Anal Chem 81(22):9522-30.
- Sequence Specific Retention Calculator (SSRCalc), http://hs2.proteome.ca/SSRCalc/SSRCalcX.html.
- Lange, V., et al. (2008). Selected reaction monitoring for quantitative proteomics: A tutorial. Mol Sys Biol 4:222.
- Kiyonami, R., et al. (2011). Increased selectivity, analytical precision, and throughput in targeted proteomics. Mol Cell Proteomics 10:M110.002931.
For Research Use Only. Not for use in diagnostic procedures.