Design of LOQ Studies for Spiked Proteins
The overall goal of our study was to test the effects of simple plasma processing methods, alone and in combination, on the LOQ, limit of detection (LOD), and assay CV for signature peptides of candidate biomarker proteins in blood. Here we define LOQ and LOD as the concentration at which the S/N of the analyte is equal to 10 and 3, respectively, with noise levels determined as described under “Experimental Procedures.” High priority was placed on defining processing methods that enabled reasonable assay throughput (tens of patient samples/day) with high assay reproducibility and specificity.
illustrates the sample processing strategy developed. The six target proteins used in all LOQ studies are shown in . Five of the proteins are non-human and therefore not found in blood. For the human target protein PSA, we obtained ELISA results for free and total PSA in nondepleted and depleted female plasma. Results confirmed that both forms of PSA were below the detection limits of the immunoassay (≤0.01 ng/ml for both species). All LOQ studies were performed in female plasma from a healthy donor. Depletion of 7 versus 12 high abundance plasma proteins was compared to determine whether depletion of the additional five proteins from the background matrix had a significant effect on the LOQ in the final MRM assay. In addition to the evaluation of depletion strategies, the effect of fractionation of plasma prior to MS on LOQ was also examined. Following reduction, carbamidomethylation, and enzymatic digestion of spiked, depleted plasma, two separate processing paths were followed (). In path 1, digested plasma was diluted to yield ~0.75 μg of total protein onto the nano-LC column, and the 13C-internal standards were added just prior to direct analysis by LC-MRM/MS. In path 2, peptides were separated by SCX, and the 13C-internal standards were added to the corresponding SCX fraction prior to analysis by LC-MRM/MS. Recovery from immunoaffinity depletion and subsequent concentration steps were evaluated by adding the target proteins to plasma prior to depletion, whereas recovery from SCX was evaluated by adding 12C-synthetic peptides prior to fractionation.
Experimental flow diagram for LOQ studies
Target proteins and their signature peptides
Peptides derived from human PSA and the five non-human proteins were selected as peptide internal standards based upon experimental observations in an LC-MS/MS analysis of each protein digested separately with trypsin. Our goal in peptide selection was to design MRM assays for two to three peptides per protein due to the fact that different peptides from the same protein can vary widely in their MS response and recovery from sample processing and because of the high potential for interference in plasma. We prejudiced our selection for moderately hydrophobic peptides that exhibit good chromatographic peak shape on reverse phase chromatography and for peptides with good ionization efficiency (as indicated by relative abundance) and high MS/MS spectral quality. For those proteins with human homologs to our spiked ones, we chose unique sequences to minimize inaccurate quantitative measurements due to the presence of endogenous forms of our signature peptides. Initially 13 13C-labeled peptides representing six proteins were synthesized. However, four of these peptides were eliminated due to early elution on reverse phase resin and/or poor chromatographic peak shape. These peptides included a third peptide derived from PSA and HRP and a second peptide derived from leptin and myoglobin. Ionization efficiency, fragmentation patterns, LC behavior, and SCX elution for all peptides were studied and optimized for the final MRM assay.
MRM Assay Configuration and Optimization
The main limitation to unbiased proteomics biomarker discovery and the targeted, candidate-based verification approaches described here is the extremely large concentration range and complexity of the proteins in the biofluids analyzed. Triple quadrupole mass spectrometers, the instruments of choice for targeted analysis, are generally considered to be low resolving instruments with resolution of only ~1000–3000. Therefore, the potential for analyte-on-analyte interference and chemical noise is greater on these instruments when the analysis is performed in a complex background such as plasma. For example, two co-eluting peptides differing in mass by up to 2 Da will have overlapping isotopic distributions, which in a targeted, MRM analysis will likely result in ion contributions from both species residing in the Q1 mass transmission window at unit resolution. Another potential source of interference is in-source fragmentation of abundant peptides where the fragment ions rather than the precursors are the source of interference. Interference at the product ion level (Q3) is caused by coincidence of a primary or secondary fragment of the precursor that has the same or nearly the same mass as the analyte transition of interest. The extent to which interference will be a significant problem depends upon the detection level one is trying to achieve for the true analyte and the abundance of the peptides giving rise to the interferences. Therefore, it is critical for MRM assay development on these instruments to select transition ions that maximize specificity and potentially minimize interferences from co-eluting species that fall within the mass windows of the analyzers. Because it is not yet possible to reliably predict the likelihood of having an interference, we approach this experimentally by selecting a minimum of three transitions per peptide to increase specificity and selectivity of the assay in plasma (). In general, transitions were chosen based upon relative abundance and m/z greater than the precursor m/z in the full scan MS/MS spectrum recorded on the 4000 Q Trap mass spectrometer. MRM assays for each signature peptide were optimized for collision energy to maximize transmission and sensitivity of the transitions being monitored. The final MRM method included 60 optimized MRMs for the six test proteins.
Following construction of the final MRM method, LC retention, MS detectability, and linearity were evaluated by titration curves in 0.1% formic acid (no biological matrix) using heavy labeled synthetic peptides only. Most peptides showed good linearity over the 0.1–500 fmol/μ
l concentration range. However, as expected, equimolar peptides showed different MS responses (Supplemental Fig. 10
). Although only the 13
C-peptides were used, we monitored the m
channel for the all-12
C-analyte to determine the percentage of the fully unincorporated 13
C for the heavy amino acids leucine and valine. Using peak area ratios for the 12
C and 13
C transitions at the high end of the titration curves, we determined that there was ≤1% residual for each 13
C-peptide standard (Supplemental Figs. 11, B–F
, and 12, B–F
). These results were consistent with full scan MS spectra recorded with high resolution on the heavy labeled peptides, LFTGHPET[13
]LEK (myoglobin) and I[13
]VGGWECamcEK (PSA) on the Orbitrap. Supplemental Figs. 11A
show the distribution of 13
C incorporation for [13
]leucine and [13
]valine, respectively. Given that the triple quadrupole instrument is operating at unit resolution, both the 13
and the 13
species will be detected in the analyte channels as noted above. The percentages of unincorporated 13
C were used to determine the amounts of internal standard to add for quantitative measurements to minimize the contribution of residual in the 12
C channels. The level of the isotopic impurity in the internal standard (which cannot be completely avoided) effectively limits the maximum useable ratio of analyte to internal standard.
Effect of Abundant Protein Depletion on LOQ
Previous studies in our laboratory with PSA spiked into depleted plasma showed improved LOQ for quantitative measurements of PSA (data not shown) as compared with reported studies for LOQ of proteins in nondepleted plasma (21
). Therefore, we evaluated the effect of depletion of plasma on LOQ in LC-MRM/MS using two different columns, the MARS hu7 (Agilent) and IgY-12 (Beckman Coulter) for depletion of 7 and 12 high abundance proteins, respectively. Calibration curves focused in the low nanogram/milliliter range were generated by adding each test protein to MARS-hu7 and IgY-12 depleted plasma at 0, 2.5, 5, 10, 25, 50, 100, 250, and 500 ng/ml. Each concentration point was digested in triplicate representing three biological replicates, and each LC-MRM/MS was performed in triplicate (technical replicates). The data are summarized in and discussed in detail for several of the proteins below.
Results of quantitative measurements for target proteins spiked into IgY-12 and MARS hu7 depleted plasma
shows the calibration curve for the INDISHTQSVSAK peptide derived from leptin in both backgrounds. The area ratio of light peptide (analyte) to heavy peptide (internal standard) was determined from the extracted ion chromatogram (XIC) of the most abundant transition for the pair, 467.2/643.8 and 468.9/646.3, respectively. These curves in both depleted plasma matrices demonstrate linearity of about 2 orders of magnitude for the concentration range tested and are representative of linearity of the other target proteins with the exception of MBP. The relative ratios of the three transitions monitored for the INDISHTQSVSAK peptide in buffer only are shown in , and provide the ideal ratios of the transition ions in the absence of interference. , are the corresponding data for this peptide obtained by spiking 25 ng/ml leptin protein into IgY-12 depleted plasma and processed as shown in (path 1). The relative ratios of the transitions agree closely with those observed in buffer indicating that no interference from the matrix is present in these channels. The most abundant transition, 467.2/643.8, was used to quantify leptin in this sample and across all concentration points. The S/N for this transition in 25 ng/ml spiked plasma was 16 (, blue), indicating that the LOQ for leptin in IgY depleted plasma is 25 ng/ml (). The precision/accuracy of this measurement across three biological replicates at this concentration was 12% CV.
Calibration curve for quantifying mouse leptin in MARS hu7 and IgY-12 depleted plasma
Extracted ion chromatograms (A–C) and MRM spectra (D–F) of transitions monitored for mouse leptin in buffer (A and D), IgY-12 (B and E), and MARS hu7 (C and F) depleted plasma
In contrast, the relative ratios of the monitored transitions for the leptin peptide in MARS hu7 depleted plasma do not correlate with the expected ratios in buffer alone. , show the overlay of the XICs and the MRM spectrum, respectively, recorded on the three transitions for the INDISH-TQSVSAK analyte peptide in MARS hu7 depleted plasma spiked with 25 ng/ml leptin. The ratio of the 467.2/586.8 transition (, red) to the 467.2/643.8 transition (, blue) is ~1.0:0.6 compared with ~0.7:1.0 in buffer alone, indicating the presence of a significant interference from the plasma matrix in this channel. The S/N of the 467.2/643.8 transition (, blue) is 7, which is approaching the limit of detection for this peptide in MARS hu7 depleted plasma. The LOQ for leptin protein spiked into MARS hu7 depleted plasma was 50 ng/ml with a CV of 26% and was based on an average S/N of 10 ().
A similar analysis of the signal to noise ratios for the remaining signature peptides derived from the other target proteins showed similar improvements in S/N, LOQ, and CV using IgY-12 depleted plasma versus the MARS depleted plasma (). With the exception of MBP, the LOQs for these target proteins were 25 ng/ml in IgY-12 depleted plasma and ≥50 ng/ml in MARS-Hu7 depleted plasma. The percent CVs of the quantitative measurements at the specified LOQ for all test proteins (, bold) were <10% for five of eight measurements in IgY-12 depleted plasma and four of eight measurements in MARS hu7 depleted plasma. No LOQ for PSA based upon the LSEPAELTDAVK peptide was reported due to the significant amount of interference observed in the most abundant MRM channel for this analyte in depleted plasma with no PSA added (see below for further discussion). The amount of total protein loaded onto the nano-LC column in all experiments was normalized for both depletion strategies by dilution of MARS-Hu7 and IgY-12 depleted plasma by 1:10 and 1:3, respectively. These dilution factors were defined by previous experiments that showed that injection of ≤1 μg of total protein onto nano-LC columns is optimum for reproducible chromatographic peak shape and robust quantitative measurements (data not shown). In effect, the removal of five additional proteins with IgY-12 versus MARS hu7 reduces the total protein concentration, allowing more of the remaining sample (including the analytes of interest) to be injected and analyzed. Based upon these results, all subsequent experiments were performed in IgY-12 depleted plasma.
Evaluation of Digestion Efficiency
Since all quantitative measurements for our target proteins are reliant upon recovery of signature peptides from proteolytic digestion, we wanted to determine and better understand the effect that digestion may have on LOQ. To test digestion efficiency, we generated calibration curves for all six target proteins with 12
C synthetic forms of our signature peptides. To directly compare the results of the 12
C-peptide curves with those generated with intact protein, we added the light signature peptides to IgY-12 depleted and digested plasma at protein concentrations equivalent to those that were used previously. Supplemental Fig. 1C
shows the comparison of the spiked light peptide and intact protein curves for the AGLCamcQTFVYG-GCamcR peptide derived from aprotinin. Both curves show excellent correlation with the expected ratio and indeed have virtually the same line shape and behavior across the concentration range. These results were typical of the rest of the signature peptides with the exception of MBP (see below). Therefore, we conclude that our digestion efficiency and reproducibility are high and do no contribute significantly to measurement variability in our studies.
Use of Multiple Peptides to Determine LOQ in Plasma
It is well known that equimolar peptides derived from the same protein can have varying MS detectability due to different ionization efficiencies. Reliable programs for predicting de novo
which peptides from a protein will give the best MS response have yet to be developed. For these reasons, multiple peptides per protein are selected for MRM assay development with the assumption that at least one will be a useful surrogate for the target protein in the final MRM assay. For the determination of LOQ for HRP and MBP, we selected two peptides from each protein. However, each of these signature peptides yielded a different LOQ for its respective protein in IgY-12 depleted plasma (). To determine the cause of these differences, we again compared calibration curves generated at equivalent concentrations with 12
C-synthetic peptides and intact protein already described above. Supplemental Figs. 8C
show the protein and 12
C-synthetic peptide calibration curves for peptides DTIVNELR and SS-DLVALSGGHTFGK derived from HRP in nonfractionated, IgY-12 depleted plasma. Curves for the identical signature peptide behaved similarly regardless of the source of analyte peptide (i.e.
spiked intact HRP followed by digestion or spiked 12
C-synthetic peptide into digested, depleted plasma). However, each signature peptide behaved differently relative to the expected ratio (Supplemental Figs. 8C
). Curves generated for spiked intact protein added to depleted plasma prior to digestion (closed triangle) and 12
C-synthetic peptides spiked into digested, depleted plasma (closed square) for the SSDLVALSGGHTFGK sequence fall close to the expected ratios (Supplemental Fig. 9C
), whereas the same curves generated for the DTIVNELR peptide (Supplemental Fig. 8C
) are lower than the expected ratios, indicating that there is an issue with this peptide that is unrelated to proteolytic digestion, e.g.
solubility. Nevertheless DTIVNELR is detected with greater efficiency than SSDLVALSGGHTFGK.
and show two different views (left versus right) of the three transitions monitored for the DTIVNELR peptide and the SSDLVALSGGHTFGK peptide of HRP, respectively. The relative ratios of the three transitions being monitored for each peptide were determined without plasma background (, and ) and compared with those signals obtained in IgY-12 depleted plasma with 25 ng/ml (, and ) and 100 ng/ml spiked HRP (, and ). For each peptide, the relative ratios of the transitions monitored with each concentration point of added HRP agree with that observed without plasma, indicating that no interference from the matrix is present. Quantification of each analyte peptide derived from HRP was based upon the most abundant transition for each signature peptide (480.3/630.3 for DTIVNELR and 492.6/790.4 for SSDLVALS-GGHTFGK), and the S/Nfor these same transitions was calculated for each concentration point shown (, blue; , blue). The lower LOQ for the DTIVNELR peptide in nonfractionated, depleted plasma as compared with that for SSDLVALSGGHTFGK of HRP is due to the about 3-fold better S/N for the former.
Extracted ion chromatograms (A–C) and MRM spectra (D–F) of transitions monitored for DTIVN-ELR derived from HRP in nonfractionated, depleted plasma
Extracted ion chromatograms (A–C) and MRM spectra (D–F) of transitions monitored for SS-DLVALSGGHTFGK derived from HRP in nonfractionated, depleted plasma
Supplemental Figs. 4C
show the protein and 12
C-synthetic peptide curves for peptides HGFLPR and YLASAS-TMDHAR derived from MBP in nonfractionated, IgY-12 depleted plasma, respectively. Both curves generated by the addition of light synthetic peptides fall above the expected peak area ratios for the range of concentrations analyzed, indicating that these signature peptides respond well in the MRM assay. However, calibration curves generated from the spike of intact protein prior to digestion are lower than the expected ratio, indicating that a problem exists either in the original protein stock concentration or in the digestion efficiency of this protein. Analysis by one-dimensional SDS-PAGE showed the purity of this protein to be ~50% (data not shown), which is likely the reason for the higher LOQ obtained for MBP.
Effect of SCX Fractionation on LOQ
Identity-based biomarker discovery has become reliant upon multidimensional fractionation at the protein and/or peptide level to improve detection of proteins of lower abundance. However, this strategy has had a negative impact on overall sample throughput with small numbers of patient samples as input and large numbers of fractions per patient to analyze. Extensive fractionation of digested plasma to improve limits of quantitation and detection for verification studies is not a practical solution because the number of cases and controls required for analysis to progress a candidate biomarker into preclinical validation is in the tens to a few hundred. To take advantage of the perceived benefit that fractionation can have on increased detection and at the same time maintain reasonable throughput, we devised a strategy for limited SCX fractionation of depleted and digested plasma that relies on the generation of SCX pools (n < 10) containing multiple signature peptides per pool.
To test our hypothesis, we separated the 0, 2.5, 5, 10, 25, 50, and 100 ng/ml spiked tryptic digests of plasma by strong cation exchange chromatography. To directly compare the effect of fractionation on LOQ with our nonfractionated results, we used the identical samples for SCX that were used to generate the calibration curves in nonfractionated plasma. Supplemental Fig. 13
shows the overlay of the elution profiles for the SCX separation of the 10 ng/ml spiked plasma digest with the separation of the signature peptides in the absence of plasma. Retention time shifts for the signature peptides were routinely monitored by injection of the standards pre- and post-limited SCX fractionation of the digested plasma samples and showed minimal change. Blank injections were performed prior to injection of each plasma sample to eliminate any 13
C-peptide carryover. Six pools of SCX fractions were generated to encompass eluting peptides in a single pool if possible and to increase analysis throughput (Supplemental Fig. 13
Analysis of all of the signature peptides for the target proteins shows that fractionation improves S/N of the analyte transitions by 10- to 40-fold, resulting in LOQ for all test proteins at or below 2.5 ng/ml in plasma (). For example, the calibration curves for the LFTGHPETLEK peptide derived from myoglobin in nonfractionated and fractionated plasma are shown in Supplemental Fig. 3D
. In SCX-fractionated plasma, calculated concentrations based upon the area ratio of analyte to IS correlate well with the expected concentrations across the range tested. In contrast, the curve generated in nonfractionated plasma has a different shape and slope with respect to the curve generated in SCX-fractionated plasma and indeed deviates significantly from the expected concentrations at the low end of the curve. This latter observation is most likely due to the presence of interferences at the lower levels of analyte. With SCX fractionation, the LOQ for the LFTGHPETLEK peptide improves to 2.5 ng/ml with 9% CV as opposed to 25 ng/ml with 16% CV in depleted, non-fractionated plasma.
Results of quantitative measurements for target proteins in SCX fractionated, depleted plasma
With SCX fractionation, the amount of peptide detected on column is as low as 100–200 amol for some peptides. The signature peptide, INDISHTQSVSAK from leptin, is particularly well behaved. shows the XICs of the three transitions monitored for the leptin peptide in nonfractionated (–C) and SCX-fractionated (–F) depleted plasma. , show no significant signal in the MRM channels at the retention time where the 13C-internal standard peptide elutes (, insets) for the 0 and 2.5 ng/ml additions of leptin, respectively, in nonfractionated depleted plasma. In (inset), the relative ratios of the transitions and the retention time correlate with that of the internal standard and show an S/N of 16 for the most abundant transition, 467.2/643.8. In the SCX-fractionated, depleted plasma, an S/N of 127 was observed for the identical transition in the 2.5 ng/ml spike of leptin () with no appreciable signal in any of the MRM channels with no leptin added (). Therefore, LOQ for leptin in depleted, SCX-fractionated plasma can be extrapolated to the picogram/milliliter range.
Extracted ion chromatograms of MRM transitions monitored for the INDISHTQSVSAK peptide derived from mouse leptin in nonfractionated (A–C) and SCX-fractionated (D–F), depleted plasma
Analysis of Interferences from Co-eluting Peptides: A Case Study of PSA
Despite the high specificity and selectivity of the MRM assay, peptides and other small molecules in the biological matrix can produce interferences in the m/z channels monitored, resulting in inaccurate quantitative measurements and overestimation of LOQ and LOD. For example, we have noted significant interference with the signature peptide LSEPAELTDAVK derived from PSA. The measured levels of free and total PSA in the female plasma we used were both below 0.01 ng/ml, so these experiments were carried out by addition of PSA protein. The calibration curves for this PSA-derived peptide provided the first indication of plasma interference (). The area ratios obtained by measuring LSE-PAELTDAVK from either exogenous PSA protein (red triangles) or spiked 12C-synthetic peptide (red squares) relative to 13C-synthetic peptide in nonfractionated plasma were both well above the expected area ratio even at high protein or peptide concentration. The other signature peptide from PSA, IVGGWECamcEK, tracks much closer to the expected area ratio, indicating little to no interference. Consistent with these observations are the high percent relative errors (>400%) calculated for the LSEPAELTDAVK peptide in non-fractionated plasma with respect to the target concentration across all concentrations of spiked protein (). To determine whether the stock protein concentration for PSA was in error, we obtained ELISA results for two additions of PSA to IgY-12 depleted female plasma, 10 and 25 ng/ml, which were consistent with our target concentrations (11.82 and 27.80 ng/ml, respectively).
Protein and 12C-synthetic peptide calibration curves for quantifying human PSA in nonfractionated, depleted plasma
The full scan MS/MS spectrum of the authentic PSA peptide () is dominated by the y9 ion formed by gas phase cleavage N-terminal to proline. Consequently the m
636.7/943.5 transition is an obvious one to monitor by MRM. We also monitored two additional transitions for this peptide ( and Supplemental Fig. 7A
). To corroborate the presence of an interference in one or more of these channels, we recorded the full scan MS/MS spectrum of m
636.7, the (M ± 2H)2+
of the LSEPAELTDAVK peptide in female plasma with and without the addition of exogenous PSA protein on the 4000 Q Trap (, respectively). A co-eluting peptide with a precursor mass residing within the mass window of Q1 is observed in the MS/MS spectrum obtained in the absence of added PSA. This interference yields a product ion at 943.4, coinciding with the most abundant transition for the authentic PSA peptide, LSEPAELTDAVK (). A co-mixture of the sequence ions for the PSA peptide and the interfering peptide is observed in the MS/MS spectrum obtained from plasma samples spiked with 100 ng/ml PSA (). Clearly the presence of a strong interference in this channel would lead to a significant overestimation of the level of PSA and the LOQ for its detection in plasma at levels at or below 100 ng/ml. Without monitoring the relative ratios of multiple transitions for this peptide, it would have been difficult to identify the presence of an interfering peptide. Knowing that interference was present at levels much greater than the desired LOQ range (low nanograms/milliliter), we constructed an assay using another PSA peptide, IVGGWECamcEK (). This peptide does not suffer significant interference from plasma in the m
transitions monitored, and we were able to develop an assay for PSA with an LOQ of ~25 ng/ml in nonfractionated plasma ().
Full scan MS/MS spectra recorded on the doubly charged ion at m/z 636.7
Interestingly the interference of the LSEPAELTDAVK peptide persists in SCX-fractionated plasma where the interfering peptide is purified together with a fraction of the authentic PSA peptide. To obtain the identity of the interfering peptide, the MS/MS spectrum of 636.7 was recorded in the SCX fraction in which the interfering peptide co-purifies with the PSA peptide (). Spectra were searched against the NCBInr database using Spectrum Mill MS Proteomics Workbench but did not produce a match. De novo interpretation of the MS/MS spectrum yielded the partial sequence VAA-CamcNLPIVR where 159 is the residual mass that must be present at the N terminus to account for the mass of the peptide. This sequence matches a tryptic peptide from α1 microglobulin (TVAACamcNLPIVR) with a modification on the N terminus. The isotopic distribution of the precursor ion as well as the y8 fragment ion from the interpreted sequence overlaps with the precursor ion and the y9 fragment ion from the PSA peptide. Therefore, the interference observed in the 636.7/943.4 channel during MRM analysis is a 13C isotope of the interpreted sequence. Synthetic peptides corresponding to the α1-microglobulin sequence as well as permutations of the N terminus are being synthesized to confirm the identity of the co-eluting peptide.
Recovery of Protein after Depletion and SCX Fractionation
To evaluate loss of protein at the immunoaffinity depletion and subsequent concentration step, we added five of the six target proteins to nondepleted plasma prior to any processing at concentrations ranging from 5000 to 2.5 ng/ml (). Aprotinin was eliminated from this analysis because of its low molecular weight with respect to the 5000 molecular weight cutoff filter that was used for the concentration step. An aliquot of the nondepleted plasma spiked with proteins at 5000 ng/ml was reserved for LC-MRM/MS following tryptic digestion, and the remaining undigested samples were depleted via IgY-12, concentrated, and digested as described under “Experimental Procedures.” Percent recoveries were determined by LC-MRM/MS of the signature peptides pre-and postdepletion (without SCX fractionation) for those spike levels at which the signature peptides could be detected (Supplemental Table 1
). To determine protein losses at the lowest protein spike levels, calibration curves were generated for the five proteins added prior to depletion of the plasma, and these samples were then depleted, fractionated by SCX, and analyzed by LC-MRM/MS as described. As anticipated, percent protein recoveries varied depending upon the specific protein. Three of the five proteins exhibited recoveries of 80–100%; PSA was recovered at ~30%. Importantly protein recoveries at the lowest spike levels were similar to those observed at 5000 ng/ml, indicating that the reproducibility of recovery is high and consistent across concentrations. MBP was not detected. By concentrating a stock solution of MBP and analyzing the retentate by gel electrophoresis, we have determined that this protein is being lost at the membrane filtration step (data not shown).
We also evaluated peptide recovery at the SCX fractionation and desalting steps separately from protein depletion by spiking eight of the well retained 12C-peptides into 100 μl of depleted and digested plasma at an equimolar amount of 50 fmol. A small aliquot was reserved for LC-MRM/MS, and the remaining sample was separated by SCX followed by desalting as described under “Experimental Procedures.” 13C-Internal standards were added to all samples at 5 fmol/μl just prior to MRM analysis (performed in triplicate). Percent recoveries ranged from >80 to 100% for the eight analyte peptides tested (data not shown).