Optimization of Sensitivity, Resolution, Throughput and Robustness in LC-MRM/MS for Protein Quantitation Kerry Nugent, Lori Ann Upton, Yixin Zhu and Christopher Loran - Michrom Bioresources, Auburn, CA
Overview
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Sahana Mollah, Christie L Hunter and Lydia Nuwaysir - AB Sciex, Foster City, CA
Results 1.0e4
NanoSpray sensitivity was compared to CaptiveSpray at nanoliter flow rates 2A
8.0e4
ElectroSpray
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2B
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20 fmol BSA 75ux150mm C18 250 nl/min
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100 fmol BSA 1x50mm C18 100 ul/min
1.8e4 Intensity, cps
Intensity, cps
Sample throughput was explored for various protein quantitation applications
NanoSpray
Intensity, cps
ElectroSpray robustness was compared to CaptiveSpray at microliter flow rates The need for resolution to minimize potential interferences was explored
Throughput
Sensitivity
This study looked at sensitivity and resolution vs throughput and robustness
Summary
1.4e4
5 fmol BSA 250 nl/min 120 min 15 BSA MRM
4A
1.6e4 1.4e4 1.2e4 1.0e4 8000.0 6000.0
Figure 4A – For very complex samples with 1000s of peptides/analytes, a long run at low nano flows will give optimum sensitivity and resolution at the expense of sample throughput.
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Introduction
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100 fmol BSA 0.2x50mm C18 8 ul/min
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Intensity, cps
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20 fmol BSA 75ux150mm C18 250 nl/min
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5 fmol BSA 1000 nl/min 30 min 15 BSA MRM
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Figure 4C – For simple samples with 10s of peptides/analytes, a very fast run at a low capillary flows will give optimum sensitivity and resolution with high sample throughput.
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Figure 4D – For targeted samples with only a few peptides/analytes, an ultra fast run at high capillary flows will give optimum sensitivity and resolution with very high sample throughput.
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Figure 4B – For less complex samples with 100s of peptides/analytes, a faster run at a high nano flows will give optimum sensitivity and resolution with better sample throughput.
5 fmol BSA 4 ul/min 10 min 15 BSA MRM
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Figure 2. A/B NanoSpray and CaptiveSpray show similar sensitivity at identical nanoflow conditions, with similiar LLOQs of 20-200 amol on the 4000 QTRAP MS (5-50 amol on the QTRAP 5500 MS – data not shown).
50 fmol BSA 20 ul/min 5 min 15 BSA MRM
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2.0e4 1.6e4 1.2e4 8000.0
Experimental
Figure 2. C/D CaptiveSpray provides 5-10x higher sensitivity than ElectroSpray when run under similar LC conditions (50mm C18 column run at comparable linear velocity from 10-30%ACN in a 4 min gradient)
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NL: 9.81E6 Base Peak MS 112009_advlc_500nl120min_AMRA_msmsde10_ 03
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1.4e5 1.3e5 1.2e5 1.1e5 1.0e5 9.0e4 8.0e4 7.0e4 6.0e4 5.0e4 4.0e4 3.0e4 2.0e4 1.0e4 0.0
5A – Run 1 of 960
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20 fmol BSA+BG in 500 ng E. coli 800 nl/min 15 min 30 BSA-BG MRM 6.0
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20 fmol BSA+BG in 500 ng E. coli 800 nl/min 15 min 30 BSA-BG MRM
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CaptiveSpray provides the sensitivity of Nanospray at nanoliter flow rates 1.0
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Figure 3 C/D – At 500nl/min, a gradient time of 40 min was required to provide enough resolution for good peptide quantitation in complex samples.
50 fmol BSA + 500 ng E. coli 500nl/min 60 min 15 BSA MRM
3D Intensity, cps
500 ng E. Coli 500nl/min 60 min EMS
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5C - Metric Plot of 960 Runs
NL: 4.64E6 Base Peak MS 112009_advlc_500nl120min_AMRA_msmsde10_ 05
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Sample throughput can be optimized to various protein quantitation applications
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CaptiveSpray provides the robustness required for quantitative applications Figure 3 E/F – At 1000nl/min, a gradient time of 20 min was required to provide enough resolution for good peptide quantitation in complex samples.
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NL: 2.59E6 Base Peak MS 112009_advlc_500nl120min_AMRB_msmsde10_ 03
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50 fmol BSA + 500 ng E. coli 1000nl/min 30 min 15 BSA MRM
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500 ng E. Coli 1000nl/min 30 min EMS
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Figure 1. A. Advance UHPLC System, B. CaptiveSpray Source and C. Q-Trap 5500 MS
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Electrospray is less sensitive than CaptiveSpray at microliter flow rates CaptiveSpray operates over a wide flow range to enable optimum resolution
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The CaptiveSpray source for AB Sciex MS instruments was found to be very robust, with little change in performance over many long term quantitation experiments (including a 240 hour sequence of 960 x 15 min runs). Although contamination of the MS was a concern prior to this work, inspection of the system before and after the 960 x 500 ng E. coli spiked digests showed minimal contamination (which was also shown in the data from runs 1 and 960 shown in this poster). CaptiveSpray is ideal for biomarker research, where the discovery phase requires the highest possible sensitivity at relatively low throughput and robustness, the validation phase requires very high sensitivity with high throughput and moderate robustness, while the clinical implementation phase requires high sensitivity, ultra high throughput and extremely robust operation.
Time, min
RT: 0.00 - 150.02 100
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60
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Figure 3 A/B – At 250nl/min, a gradient time of 80 min was required to provide enough resolution for good peptide quantitation in complex samples.
50 fmol BSA + 500 ng E. coli 250nl/min 120 min 15 BSA MRM
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500 ng E. Coli 250nl/min 120 min EMS
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1C
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Resolution Intensity, cps
A CaptiveSpray source from Michrom was developed for triple quadrupole and QTRAP MS systems from AB SCIEX. Several quantitative proteomics applications were tested using a capLC-CSI/MS, nanoLC-NSI/MS and LC-ESI/MS to determine optimum LC-MRM/MS conditions for each application and understand the tradeoffs for robustness/throughput vs. sensitivity/resolution. An E. coli lysate digest was used as a complex matrix, with standard protein digests added at known concentrations and monitored using MRM-MS, on either a 4000 QTRAP® MS or a QTRAP® 5500 MS. Robustness testing consisted of 100-1000 injections run on a single column and spray tip combination. Sensitivity experiments used the same samples on the multiple configurations and compared LLOQ quantitative results. A range of LCMS conditions were then explored to interrogate different experimental scenarios that may be encountered in research or clinical settings. 1B
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LCMS is a key technology for the analysis of biomolecules. Pharmaceutical scientists utilize analytical HPLC coupled with ElectroSpray (ESI) MS for both qualitative and quantitative analysis of drugs and metabolites, as this technique provides high throughput and robust operation. Protein chemists utilize nanoLC coupled with NanoSpray (NSI) MS for the identification and characterization of proteins, as this technique provides high sensitivity and high resolution. With the recent interest in proteins as biomarkers, the field of quantitative proteomics requires the sensitivity of nanoLC-NSI/MS, with the robustness of LC-ESI/MS. This study utilizes an innovative source technology (CaptiveSpray-CSI) which bridges the gap between high flow and nano flow and provides an excellent solution for maximizing robustness and throughput while still maintaining good sensitivity.
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This study shows that CaptiveSpray ionization is a great compliment to both NanoSpray ionization and ElectroSpray ionization for quantitative proteomics. CaptiveSpray, when coupled with a nano/capillary UHPLC system and Q-Trap MS, provides the sensitivity of nanoLC-NSI/MS with the robustness of LC-ESI/MS, both of which are important for protein quantitation applications. The CaptiveSpray source worked well on a variety of MS systems (although like NanoSpray, sensitivity was 510x higher on the newer QTRAP 5500 MS used by the authors from AB Sciex vs the older 4000 QTRAP MS used by the authors from Michrom) and was also compatible with a variety of LC systems (Eksigent nanoLC, Advance nano/cap UHPLC and Paradigm micro/analytical MDLC) over a broad flow range (200 nl/min to 20 ul/min).
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Figure 5. A/B To test the robustness of CaptiveSpray, a sample containing bovine serum albumin, beta-galactosidase and an E. coli lysate was digested with trypsin and 20 ul aliquots of the digest were loaded into a 96 well plate on the autosampler. 960 consecutive 15 min runs (800 nl/min on a 0.1x150mm C18 column) were performed to test robustness, and the performance of run 1 and run 960 were very simiar. Figure 5. C A metric plot of the peak areas from three representative BSA peptides showed very reproducible performance (CVs
