Quality Control Measures for Routine, High-Throughput Targeted Protein Quantitation Using Tandem Capillary Column Separation

Transcription

1 Quality Control Measures for Routine, High-Throughput Targeted Protein Quantitation Using Tandem Capillary Column Separation Sebastien Gallien 1, Elodie Duriez 1, Amol Prakash, Scott Peterman, Andreas Huhmer 3, Evert-Jan Sneekes 4, Remco Swart 4, and Bruno Domon 1 1 Luxembourg Clinical Proteomics Center, Strassen, Luxembourg; Thermo Fisher Scientific, Cambridge, MA, USA; 3 Thermo Fisher Scientific, San Jose, CA, USA; 4 Thermo Fisher Scientific, Amsterdam, The Netherlands

2 Overview Purpose: Develop a targeted, tandem LC method for high-throughput complex targeted peptide quantitation Methods: A peptide retention time calibration (PRTC) kit was used to characterize target peptide elution profiles using a constant LC gradient as well as different LC gradients targeting different peptides per experiment. The measured PRTC peptide response was also used for system QC. Results: The targeted, tandem LC method used for peptide quantitation increased target peptide separation and measured response and quantitation while maintaining excellent reproducibility. The PRTC kit facilitated a QC routine for normalization to show over 9% of the targeted peptides targeted within % variance for AUC values and retention times. Introduction Traditional LC/MS-based targeted peptide quantitation assays incorporate large numbers of proteotypic peptides to determine protein expression levels. Experimental methods use generic LC methods to attempt to facilitate all peptides, similar to those employed in discovery experiments despite the majority of the targeted peptides having similar physiochemical properties. Attempts to increase peak capacity employ extremely long gradients, which greatly reduce throughput, or higher flow rates on microbore columns requiring greater sample to achieve similar detection limits. To overcome these limitations, tandem LC experiments have been implemented. The two column arrangement facilitates greater throughput by staggering the sample loading/washing on one column while performing analytical gradient/data collection on the second. Previous analytical methods incorporating tandem LC approaches focused only on addressing throughput by employing similar, LC columns and gradients. Generic, linear gradients compromise maximum LC separation and sensitivity but are used due to a lack of QC protocols amenable across any LC parameter. Our approach is to incorporate a Peptide Retention Time Calibration (PRTC) kit to characterize the LC and MS parameters and provide for internal QC protocols for targeted LC and MS experiments using a tandem LC arrangement. Methods Sample Preparation An expanded PRTC kit composed of the commercially available Thermo Scientific Pierce Peptide Retention Time Calibration Mixture and a mixture of 17 additional wellcharacterized synthetic peptides was spiked into each sample at an equal molar amount. The targeted peptide list was comprised of 37 Thermo Scientific heavylabeled synthetic peptides mixed at equal molar amounts for all analyses. The PRTC kit and targeted, heavy-labeled peptides were spiked into a constant background matrix comprised of ng/µl digested urine proteins. A separate sample was prepared for each column and experiment. Liquid Chromatography All experiments were performed in tandem LC mode using a Thermo Scientific Dionex UltiMate 3 RSLCnano liquid chromatograph equipped with two sets of UHPLC pumps. Each pump used common LC solvents (A:.1% formic acid and B:.1% formic acid in MeCN) and maintained a flow rate of 3 nl/min. Each system used a Thermo Scientific Dionex Acclaim PepMap x.7 mm i.d. trap column and an Acclaim PepMap RSLC 1 x.7 mm analytical column. Two sets of tandem LC experiments were performed. The first used identical LC methods on both columns and the second used different LC methods on each column to better separate hydrophilic (column 1) and hydrophobic (column ) peptides. Mass Spectrometry All experiments were acquired using the Thermo Scientific Q Exactive hybrid quadrupole-orbitrap mass spectrometer. All data was acquired using high-resolution, accurate-mass (HR/AM) full-scan MS m/z ) and targeted MS/MS events on the 37 precursors as a function of retention time m/z ). Data Analysis Product ion spectra were searched against a limited database in Thermo Scientific Proteome Discoverer software for verification. All targeted acquisition methods, data processing, and verification was performed in Thermo Scientific Pinpoint software version 1.3. Targeted peptide quantitation was performed at the MS level using ± ppm mass tolerance for data extraction. Quality Control Measures for Routine, High-Throughput Targeted Protein Quantitation Using Tandem Capillary Column Separation

3 Results The present example targeted a large list of peptides from over biologically relevant urine proteins. The goal of the research was to build robust LC/MS methods providing quantitative assessment for groups of urine proteins. However the dramatic overlap in peptide hydrophobicities (Figure 1) reduced the targeted acquisition duty cycle for any one peptide. FIGURE 1. Distribution of calculated hydrophobicity factors for a panel of targeted peptides from urine bladder cancer proteins. The list contains 37 peptides used as surrogate biomarkers for over 137 proteins. Hydrophobicity factors were calculated using the Sequence Specific Retention Calculator (SSRCalc) Figure 3 shows the comparative results Method B. The two insets show the repr two columns. Mapping of the PRTC pep used across each column and provides pump and column, and mass spectrome composed of tryptic peptides, the perfor targeted peptides. The additional benefi hydrophobicity factors for the kit that ex PRTC peptides were used to correlate t and enabled the linear response correla retention time values. More than 9% o %. Incorporation of the PRTC kit facil gradients or columns # of Peptides Hydrophobicity Factor Over 7% of the tryptic peptides have calculated hydrophobicity factors in the range of 36 and very few have factors in the extremes, yet a majority of LC methods use gradients running from 3% 4% organic over a set period of time (typically.7% per minute). This results in wasted data acquisition time without addressing separation compromises, throughput and sensitivity. We incorporated tandem LC and utilized the two column arrangement to increase chromatographic separation and sensitivity and maintain throughput for complex targeted peptide quantitation methods. Figure shows the tandem LC experiment. Identical LC columns and solvents were used for the present study, but different combinations of columns and/or solvents will be employed to create better separation. The two different methods were used to demonstrate that even modest changes in the solvent profiles result in significant changes. The first experiment used identical methods, while the second incorporated two different LC solvent profiles and targeted the more hydrophilic range (Method C) and hydrophobic (Method D) peptide. Both methods benefitted from the tandem LC approach, reducing the overall analysis time from 7 minutes for each column to 3 minutes. The primary difference between the methods was the starting concentration of MeCN (% for Methods A, B, and C, and 9% for D) and the ending concentration (3% for A and B, % for C, and 9% for D). The length of the analytical portion of the experiment remained constant at 3 minutes. FIGURE. Schematic of tandem LC column approach used for targeted quantitation experiments. The two methods show the loading, analytical, and washing portion of the experiment for the tandem LC events. The solid line represents the data acquisition portion of the method. % MeCN % MeCN Time (min) Time (min) The use of tandem LC doubled the thro capacities. To expand on the separation different methods, the second experime starting concentration of MeCN but incre to maximize separation for hydrophobic (Figure 4) To evaluate the targeted pept used (average peak width) covering the hydrophobicity factors (6-). The avera Method C and 7.1 for /B with m eluting. The greater number of co-elutin that can be simultaneously quantified us average AUC value increased around 1 Method D was used to preferentially res between -36) during the analytical gra peptides tend to elute during the initial 6 spreading of the elution profiles for the t Even though the LC method for D was i perfectly to the targeted peptides and d Thermo Scientific Poster Note PN6363_E 6/1S 3

4 Figure 3 shows the comparative results from the first experiment using and Method B. The two insets show the reproducible chromatographic response across the two columns. Mapping of the PRTC peptides provides retention time landmarks to be used across each column and provides a measure of QC for the autosampler, LC pump and column, and mass spectrometer performance. Since the PRTC kit is composed of tryptic peptides, the performance is expected to mimic that of the targeted peptides. The additional benefit of the PRTC peptides is the wider range of hydrophobicity factors for the kit that extends past that of the targeted peptides. The PRTC peptides were used to correlate the expected retention time across the columns and enabled the linear response correlation to be applied to confirm targeted peptide retention time values. More than 9% of the targeted peptides had %CV s less than %. Incorporation of the PRTC kit facilitated predictive scheduling across different LC gradients or columns. FIGURE 3. Comparative retention time mapping for the PRTC (filled diamonds) and targeted peptides (open diamonds) using the identical methods across two columns. The inset figures show the measured retention time as a function of calculated hydrophobicity factors. The main figure shows the correlated retention time for each PRTC and targeted peptide across each column. The histogram shows the measured retention time error across the two columns. FIGURE 4. Comparative retention tim diamonds) and targeted peptides (op Method D (b) as a function of the calc (a) Method C (b) Method D Method B PRTC y =.41x.1786 R =.989 Targeted Peptides y =.93x 4.86 R =.987 Method B PRTC y =.47x.387 R =.99 Targeted Peptides y =.366x.483 R =.941 Cross-column Correlation y =.9993x +.83 R =.9974 The use of tandem LC doubled the throughput while maintaining adequate peak capacities. To expand on the separation capabilities and test the incorporation of two different methods, the second experiment was performed. Method C used a similar starting concentration of MeCN but increased only to %. The goal of Method C was to maximize separation for hydrophobic peptides compared to Methods A and B. (Figure 4) To evaluate the targeted peptide elution density, 3-second wide bins were used (average peak width) covering the measured retention times for the range of hydrophobicity factors (6-). The average frequency count per bin was.3 using Method C and 7.1 for /B with multiple time bins having over 13 peptides coeluting. The greater number of co-eluting peptides reduces the number of peptides that can be simultaneously quantified using a targeted method. In addition, the average AUC value increased around 1.4-fold over that measured for Methods A/B. Method D was used to preferentially resolve more hydrophobic peptides (HF values between -36) during the analytical gradient. Figure 4b shows that the hydrophilic peptides tend to elute during the initial 6 minute hold at 9% then facilitate a more even spreading of the elution profiles for the targeted peptides compared to Methods A/B. Even though the LC method for D was initially static, the PRTC peptides mapped perfectly to the targeted peptides and determined the onset of gradient elution at those FIGURE. Retention time correlation Methods C and D. The PRTC peptides elution (RT > 1. min) and the linear p had a calculated regression value of 4 Quality Control Measures for Routine, High-Throughput Targeted Protein Quantitation Using Tandem Capillary Column Separation

5 sing and response across the e landmarks to be utosampler, LC PRTC kit is ic that of the he wider range of ted peptides. The across the columns m targeted peptide %CV s less than across different LC dequate peak corporation of two C used a similar al of Method C was hods A and B. ond wide bins were for the range of was.3 using er 13 peptides cober of peptides addition, the for Methods A/B. ptides (HF values t the hydrophilic cilitate a more even d to Methods A/B. ptides mapped dient elution at those FIGURE 4. Comparative retention time mapping for the PRTC peptides (filled diamonds) and targeted peptides (open diamonds) using Method C (a) and Method D (b) as a function of the calculated hydrophobicity factors Measured Retention Time (min) Measured Retention Time (min) peptides (both PRTC and targeted) at 17, as it did for the peptides in Method C where the onset of the gradient elution was observed to be at HF = 13 for both sets of peptides. The upper end of the LC elution profile also bracketed the most hydrophobic peptides and defined the chromatographic stop time. The two different experiments demonstrated increased separation efficiencies for different peptides. A large portion of the targeted peptides was overlapping. The extended PRTC kit facilitated both AUC value as well as retention time normalization. Using the PRTC peptides as retention time landmarks across the two different LC methods scheduled, targeted methods could be generated using Pinpoint software. The approach took the measured PRTC peptide retention times for both methods and determined the correlating equation. Once the equation was established, the measured retention times from Method C were used to predict the Method D retention times. The results are shown in Figure. Over 8% of the 37 targeted peptides had retention time errors less than 7% despite the difference in LC gradients. In addition, Method D also demonstrated greater chromatographic separation for the complex portion of the targeted peptides with hydrophobicity factors ranging from 36. Figure 6 shows the retention time binning for 3 targeted peptides falling into this hydrophobicity factor range. Retention Time Method D Calculated Hydrophobicity Factor (a) Method C (b) Method D Calculated Hydrophobicity Factor FIGURE. Retention time correlation for the PRTC and targeted peptides across Methods C and D. The PRTC peptides were used to establish the onset of elution (RT > 1. min) and the linear portions of the gradient. The linear equation had a calculated regression value of.988 covering a HF range of 1 to Retention Time Method C Note that the bin population for Method peptides analyzed using. Also the LC gradient. The binning profile also less complex background as well. Comp targeted peptides provides further suppo increases dramatically as a function of r approximately 1.4-fold over the portion o FIGURE 6. Comparative retention tim containing calculated hydrophobicity and Method D. Peptide Frequency AUC Ratio [Method D:] Method D Retentio FIGURE 7. Plot of AUC ratios for the 3 as a function of the measur 1 Measured Reference 1. Sequence Specific Retention Calc Thermo Scientific Poster Note PN6363_E 6/1S

6 peptides (filled hod C (a) and ctors Note that the bin population for Method D is more evenly spread than that for the same peptides analyzed using. Also, the peptides elute over a greater portion of the LC gradient. The binning profile also represents non-targeted peptides resulting in less complex background as well. Comparison of the measured AUC values for all targeted peptides provides further support. Figure 7 shows the AUC ratio [D:A] increases dramatically as a function of retention time and levels off to ratios approximately 1.4-fold over the portion of the chromatogram targeted in Method D. FIGURE 6. Comparative retention time binning results for targeted peptides containing calculated hydrophobicity factors between -36 between and Method D. Peptide Frequency Method D Retention Time Windows (min) FIGURE 7. Plot of AUC ratios for the 37 targeted peptides for Method D vs. as a function of the measured retention time AUC Ratio [Method D:] Measured Retention Time (Method D) ed peptides across the onset of The linear equation e of 1 to. Conclusion Incorporation of the PRTC kit increases the capabilities of tandem LC experiments for targeted protein quantitation: Increases throughput while maintaining low flow rates and longer gradients necessary to maintain peak capacities. Provides direct retention-time prediction using all types of LC methods on all C18-based LC columns. Facilitates implementation of different LC experiments using tandem LC to maximize sensitivity for different sets of targeted peptides. Facilitates internal RT and QC landmarks for each experiment for normalization based on the breadth of the hydrophobicity factors for the PRTC peptides. Reference 1. Sequence Specific Retention Calculator (SSRCalc), All trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. 6 Quality Control Measures for Routine, High-Throughput Targeted Protein Quantitation Using Tandem Capillary Column Separation

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