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

Results and Discussion

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.

88321-v15-Fig1-Peptide-Retention Figure 1. Chromatographic analysis of the calibration mixture. The Pierce Peptide Retention Time Calibration Mixture (250fmol) was analyzed in duplicate. Panel A: Analysis using the Orbitrap* LTQ XL Mass Spectrometer and a self-packed column (75μm × 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. Panel B: Analysis using the TSQ Vantage Mass Spectrometer and a Thermo Scientific Hypersil Gold C18 Column (1.0 × 150mm) with a 1.0% per minute gradient at 120μL per minute. The numbered peaks correspond to the calibration peptides in Table 1.

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, 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)
1 SSAAPPPPPR 493.77 7.56
2 GISNEGQNASIK 613.32 15.50
3 HVLTSIGEK 499.28 15.52
4 DIPVPKPK 451.28 17.65
5 IGDYAGIK 422.73 19.15
6 TASEFDSAIAQDK 695.83 25.88
7 SAAGAFGPELSR 586.80 25.24
8 ELGQSGVDTYLQTK 773.89 28.37
9 GLILVGGYGTR 558.32 32.18
11 SFANQPLEVVYSK 745.39 34.96
12 LTILEELR 498.80 37.30
13 NGFILDGFPR 573.30 40.42
14 ELASGLSFPVGFK 679.37 41.18
15 LSSEAPALFQFDLK 787.42 46.66
88321-v15-Fig2-Peptide-Retention Figure 2. Retention time prediction with hydrophobicity. The Pierce Peptide Retention Time Calibration Mixture sequences and chromatographic data from Figure 1B were used to calculate the retention time-to-hydrophobicity relationship in Pinpoint* 1.1 Software. The observed retention times of 29 tryptic peptides from bovine serum albumin on a TSQ Vantage Mass Spectrometer were plotted against the predicted retention times.

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).

88321-v15-Fig3-Peptide-Retention Figure 3. Prediction of relative peptide retention times between LC-MS systems. The Pierce Peptide Retention Time Calibration Mixture was analyzed using LTQ Orbitrap XL and TSQ Vantage Mass Spectrometers. Enzymatic digests from lysed 293T cells treated with 5μg/mL insulin for 30 minutes were analyzed on an LTQ Orbitrap XL Mass Spectrometer. Search results from Thermo Scientific Proteome Discoverer 1.1 Software and the calibrant results from both platforms were imported into Pinpoint 1.1 to develop scheduled selective reaction monitoring assays for 478 peptides in 35 proteins. Retention times observed on the TSQ Vantage Instrument were plotted against the retention times observed on the Orbitrap XL Instrument.

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.

Cited References
  1. 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.
  2. 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.
  3. Sequence Specific Retention Calculator (SSRCalc),
  4. Lange, V., et al. (2008). Selected reaction monitoring for quantitative proteomics: A tutorial. Mol Sys Biol 4:222.
  5. Kiyonami, R., et al. (2011). Increased selectivity, analytical precision, and throughput in targeted proteomics. Mol Cell Proteomics 10:M110.002931.