Improving the Sensitivity of Protein Quantification by Immunoaffinity Liquid Chromatography—Triple Quadrupole Mass Spectrometry Using an Iterative Transition Summing Technique

The desire to reach ever-diminishing lower limits of quantification (LLOQ) to probe changes in low abundance protein targets has led to enormous progress in sample preparation and liquid chromatography–tandem mass spectrometry (LC-MS/MS) instrumentation. To maximize signal and reduce noise, many approaches have been employed, including specific immunoaffinity (IA) enrichment and reducing the LC flow to the nanoflow (nLC) level; however, additional sensitivity gains may still be required. Recently, a technique termed “echo summing” has been described for small-molecular-weight analytes on a triple quadrupole (QqQ) MS where multiple iterations of the same, single selected reaction monitoring (SRM) transition are collected, summed, and integrated, yielding significant analyte dependent signal-to-noise (S/N) improvements. Herein, the direct applicability of echo summing to protein quantification by sequential IA combined with nLC-MS/MS (IA-nLC-MS/MS) is described for a beta nerve growth factor (NGF) and a soluble asialoglycoprotein receptor (sASGPR) assay from human serum. Five iterations of echo summing outperformed traditional collection in relative average accuracy (−1.5 ± 7.7 vs −41.7 ± 10.7% bias) and precision (7.8 vs 18.4% coefficient of variation (CV)) of the low-end quality control (QC) sample (N = 4) for NGF and improved functional sensitivity of serially diluted serum QC samples (N = 5 each population) approximately 2-fold (1.96 and 2.00-fold) for two peptides of sASGPR. Echo summing also extended the minimum quantifiable QC level for sASGPR 4-fold lower. Similar gains are believed to be achievable for most protein IA-nLC-MS/MS assays.


Additional LC and MS Operating Parameters
For analysis of NGF, 85µL of sample volume resulting from protein IA (R&D / AF256, biotinylated in-house) followed by alkylation, reduction, and tryptic digestion in the presence of 0.9 fmol/µL of stable isotope labelled (SIL) IDTACVCVLSR^ (^ = 13 C6, 15 N4) was injected onto the LC system described in the publication operating in an online peptide IA configuration [12][13][14] .
The eluate from the nLC column was introduced into a TSQ Altis QqQ (Thermo Fisher Scientific; Waltham, MA) outfitted with an EASY-Spray nLC ESI source operating under the following source conditions for all analysis: capillary voltage +3000V, ion transfer tube temperature 300 C, CID Gas 2 mTorr, Source Fragmentation 0V, Chromatographic Peak Width 20 sec, chromatographic filter on.For analysis of NGF, QqQ resolution was set to Q1 resolution (FWHM) 0.7 Da, Q3 resolution (FWHM) 0.4 Da.For analysis of sASGPR, QqQ resolution was set to Q1 resolution (FWHM) 0.4 Da, Q3 resolution (FWHM) 0.2 Da.

S3
Dwell Time Theory Predicted Sensitivity Gains Associated with Echo Summing      Table S5.Comparison of the calibration curve statistics for traditional vs echo summing transition collection for the quantification of sASGPR using QFVSDLR peptide.Values outside of acceptance criteria are bolded red and denoted by an asterisk.Calibrators below 31.25 pg/mL could not be quantified and are excluded from data reporting.

Functional S/N vs n for NGF Echo Summing
Table S6.Comparison of the calibration curve statistics for traditional vs echo summing transition collection for the quantification of sASGPR using SLESQLEK peptide.Values outside of acceptance criteria are bolded red and denoted by an asterisk.Calibrators below 31.25 pg/mL could not be quantified and are excluded from data reporting.
Table S7.Comparison of the precision (expressed as the CV in the measured sASGPR concentration using QFVSDLR peptide) and accuracy (expressed as the mean bias ± SD from expected sASGPR concentration using QFVSDLR peptide) across 3 replicates of 10 or 20uL of undiluted serum collected by traditional vs echo summing transition collection.Individual values outside of acceptance criteria are bolded and denoted by an asterisk while average values are bolded red.
Table S8.Comparison of the precision (expressed as the CV in the measured sASGPR concentration using SLESQLEK peptide) and accuracy (expressed as the mean bias ± SD from expected sASGPR concentration using SLESQLEK peptide) across 3 replicates of 10 or 20uL of undiluted serum collected by traditional vs echo summing transition collection.Individual values outside of acceptance criteria are bolded and denoted by an asterisk while average values are bolded red.

Figure S1 .
Figure S1.Increases in S/N due to echo summing n iterations are predicted by dwell time theory to be offset by the decrease in S/N resulting from the faster scan speed or dt

Figure S2 .
Figure S2.Comparison of the calibration curve plots for traditional (A) vs echo summing (B) transition collection for the quantification of NGF.Calibrators that fail to meet the accuracy acceptance criteria of ± 20% are excluded from the calibration curve and denoted by open circles in the graph.The lower calibration range is determined by the lowest calibrator for which both replicates meet the accuracy acceptance criteria.

Figure S3 .
Figure S3.Extracted chromatograms of the light and SIL y7 transition of NGF collected traditionally as a single transition (A) collected as five individual echo transitions (B) and the echo summing of the five individual echo transitions from panel B (C). Signal counts presented are mean values generated from replicate runs (N=5 for matrix blank, N=2 for CAL2).Chromatograms chosen for depiction are those which have the highest correlation to the mean values.

Figure S4 .
Figure S4.Comparison of the calibration curve plots for traditional (A) vs echo summing (B) transition collection for the quantification of sASGPR using QFVSDLR peptide.Calibrators that fail to meet the accuracy acceptance criteria of ± 20% are excluded from the calibration curve and denoted by open circles in the graph.The lower calibration range is determined by the lowest calibrator for which both replicates meet the accuracy acceptance criteria.

Figure S5 .
Figure S5.Comparison of the calibration curve plots for traditional (A) vs echo summing (B) transition collection for the quantification of sASGPR using SLESQLEK peptide.Calibrators that fail to meet the accuracy acceptance criteria of ± 20% are excluded from the calibration curve and denoted by open circles in the graph.The lower calibration range is determined by the lowest calibrator for which both replicates meet the accuracy acceptance criteria.

Table S4 .
Experimental parameters utilized and data generated when assessing echo summing performance of the y7 transition of NGF with additional n values in a fixed cycle time of 0.65 seconds.