Multiplexed Quantitation of Endogenous Proteins in Dried Blood Spots by Multiple Reaction Monitoring - Mass Spectrometry

Dried blood spot (DBS) sampling, coupled with multiple reaction monitoring mass spectrometry (MRM-MS), is a well-established approach for quantifying a wide range of small molecule biomarkers and drugs. This sampling procedure is simpler and less-invasive than those required for traditional plasma or serum samples enabling collection by minimally trained personnel. Many analytes are stable in the DBS format without refrigeration, which reduces the cost and logistical challenges of sample collection in remote locations. These advantages make DBS sample collection desirable for advancing personalized medicine through population-wide biomarker screening. Here we expand this technology by demonstrating the first multiplexed method for the quantitation of endogenous proteins in DBS samples. A panel of 60 abundant proteins in human blood was targeted by monitoring proteotypic tryptic peptides and their stable isotope-labeled analogs by MRM. Linear calibration curves were obtained for 40 of the 65 peptide targets demonstrating multiple proteins can be quantitatively extracted from DBS collection cards. The method was also highly reproducible with a coefficient of variation of <15% for all 40 peptides. Overall, this assay quantified 37 proteins spanning a range of more than four orders of magnitude in concentration within a single 25 min LC/MRM-MS analysis. The protein abundances of the 33 proteins quantified in matching DBS and whole blood samples showed an excellent correlation, with a slope of 0.96 and an R2 value of 0.97. Furthermore, the measured concentrations for 80% of the proteins were stable for at least 10 days when stored at −20 °C, 4 °C and 37 °C. This work represents an important first step in evaluating the integration of DBS sampling with highly-multiplexed MRM for quantitation of endogenous proteins. Dried blood spot (DBS) sampling, coupled with multiple reaction monitoring mass spectrometry (MRM-MS), is a well-established approach for quantifying a wide range of small molecule biomarkers and drugs. This sampling procedure is simpler and less-invasive than those required for traditional plasma or serum samples enabling collection by minimally trained personnel. Many analytes are stable in the DBS format without refrigeration, which reduces the cost and logistical challenges of sample collection in remote locations. These advantages make DBS sample collection desirable for advancing personalized medicine through population-wide biomarker screening. Here we expand this technology by demonstrating the first multiplexed method for the quantitation of endogenous proteins in DBS samples. A panel of 60 abundant proteins in human blood was targeted by monitoring proteotypic tryptic peptides and their stable isotope-labeled analogs by MRM. Linear calibration curves were obtained for 40 of the 65 peptide targets demonstrating multiple proteins can be quantitatively extracted from DBS collection cards. The method was also highly reproducible with a coefficient of variation of <15% for all 40 peptides. Overall, this assay quantified 37 proteins spanning a range of more than four orders of magnitude in concentration within a single 25 min LC/MRM-MS analysis. The protein abundances of the 33 proteins quantified in matching DBS and whole blood samples showed an excellent correlation, with a slope of 0.96 and an R2 value of 0.97. Furthermore, the measured concentrations for 80% of the proteins were stable for at least 10 days when stored at −20 °C, 4 °C and 37 °C. This work represents an important first step in evaluating the integration of DBS sampling with highly-multiplexed MRM for quantitation of endogenous proteins. Dried Blood Spot (DBS) 1The abbreviations used are:DBSdried blood spotIEFisoelectric focusingHPLChigh performance liquid chromatographySISstable isotope-labeled internal standardsMRMmultiple reaction monitoringLDRlinear dynamic rangeWBwhole blood. 1The abbreviations used are:DBSdried blood spotIEFisoelectric focusingHPLChigh performance liquid chromatographySISstable isotope-labeled internal standardsMRMmultiple reaction monitoringLDRlinear dynamic rangeWBwhole blood. samples have many advantages over blood serum or plasma and are the preferred clinical sample for newborn screening for metabolic diseases (1Chace D.H. Kalas T.A. Naylor E.W. Use of tandem mass spectrometry for multianalyte screening of dried blood specimens from newborns.Clin. Chem. 2003; 49: 1797-1817Crossref PubMed Scopus (502) Google Scholar, 2Sahai I. Marsden D. Newborn Screening.Crit. Rev. Clin. Lab. Sci. 2009; 46: 55-82Crossref PubMed Scopus (41) Google Scholar). These samples are collected by pricking a newborn's heel and spotting a drop of blood onto specially designed filter paper collection cards. Samples are then dried under ambient conditions and are usually stored with desiccant at room temperature until analysis. This sampling procedure is simpler and less invasive then intravenous blood draws, which require a trained phlebotomist. Not surprisingly, the majority of adult patients prefer the small lancet used in finger-prick blood sampling methods to the larger needles used in intravenous blood draws (3Merton G. Jones K. Lee M. Johnston A. Holt D.W. Accuracy of cyclosporin measurements made in capillary blood samples obtained by skin puncture.Therapeutic Drug Monitoring. 2000; 22: 594-598Crossref PubMed Scopus (35) Google Scholar, 4Woods K. Douketis J.D. Schnurr T. Kinnon K. Powers P. Crowther M.A. Patient preferences for capillary vs. venous INR determination in an anticoagulation clinic: a randomized controlled trial.Thrombosis Res. 2004; 114: 161-165Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Unlike plasma or serum samples, which consume ≥250 μl of blood and must be centrifuged within an hour of collection, DBS samples can be prepared using a volume of only 10 μl, and do not require any specialized equipment at the collection site (5Burnett J.E. Dried blood spot sampling: practical considerations and recommendation for use with preclinical studies.Bioanalysis. 2011; 3: 1099-1107Crossref PubMed Scopus (45) Google Scholar). The simplicity and reduced safety risks associated with DBS sampling enables collection by minimally trained staff or by the patients themselves. In addition, many analytes are stable in the DBS format at room temperature, reducing sample transportation and storage costs, as well as the impact on the environment. Finally, DBS samples are safer to transport and are considered exempt from dangerous goods regulations (6Federal Register. 49 CFR part 173 Shippers General Requirements for Shipments and packaging. 2010; (Section 173.134(b) http://ecfr.gpoaccess.gov/cgi/t/text/text-idx?c=ecfr&sid=8662f082afd4c81e20b32e6f54361a73&rgn=div8&view=text&node=49:2.1. 1.3.9.4.25.13&idno=49)Google Scholar, 7World Health Organization. Guidance on regulations for the transport of infectious substances. Geneva, 2011–2012Google Scholar). These advantages make DBS sampling very attractive for advancing personalized medicine and population-based biomarker research (8McDade T.W. Williams S. Snodgrass J.J. What a drop can do: dried blood spots as a minimally invasive method for integrating biomarkers into population-based research.Demography. 2007; 44: 899-925Crossref PubMed Scopus (503) Google Scholar). dried blood spot isoelectric focusing high performance liquid chromatography stable isotope-labeled internal standards multiple reaction monitoring linear dynamic range whole blood. dried blood spot isoelectric focusing high performance liquid chromatography stable isotope-labeled internal standards multiple reaction monitoring linear dynamic range whole blood. Numerous biomolecular targets covering genomics, metabolomics, and proteomics applications have been quantified in DBS samples using a wide array of analytical techniques (9Tanna S. Lawson G. Analytical methods used in conjunction with dried blood spots.Anal. Meth. 2011; 3: 1709-1718Crossref Scopus (42) Google Scholar). The most common clinical application of DBS sampling is screening newborns for metabolomics disorders by targeting small molecule biomarkers. screening on and of which required a assay for of I. Marsden D. Newborn Screening.Crit. Rev. Clin. Lab. Sci. 2009; 46: 55-82Crossref PubMed Scopus (41) Google Scholar). the and cost required to assay the of diseases be to only a In addition, a single biomarker the to a is the only protein is targeted in DBS samples, and screening is by liquid chromatography or isoelectric focusing methods I. Marsden D. Newborn Screening.Crit. Rev. Clin. Lab. Sci. 2009; 46: 55-82Crossref PubMed Scopus (41) Google Scholar). to screening are and are not to with protein In newborn screening challenges associated with small molecule were with the of multiple reaction monitoring mass spectrometry into the clinical (1Chace D.H. Kalas T.A. Naylor E.W. Use of tandem mass spectrometry for multianalyte screening of dried blood specimens from newborns.Clin. Chem. 2003; 49: 1797-1817Crossref PubMed Scopus (502) Google Scholar, mass a method for with for screening for of PubMed Scopus Google Scholar). The of MRM enables of analytes to be a single to the of highly multiplexed The of stable isotope-labeled internal standards enables the of highly reproducible a of at is common for small molecule targets and to be by at a cost of sample M. newborn screening of metabolic disorders by tandem mass clinical and PubMed Scopus Google Scholar). of the screening panel to biomarkers on an cost less than In to newborn DBS sampling with is also in Dried blood spot sampling in with for of small 2010; PubMed Scopus Google Scholar, The of dried blood spot samples using liquid chromatography tandem mass 2011; 44: PubMed Scopus Google Scholar). Here the collection of blood sampling from reducing the of required to preclinical and (5Burnett J.E. Dried blood spot sampling: practical considerations and recommendation for use with preclinical studies.Bioanalysis. 2011; 3: 1099-1107Crossref PubMed Scopus (45) Google Scholar). the use of DBS samples in for small molecule have been of using this technology for protein targets The of dried blood spot samples using liquid chromatography tandem mass 2011; 44: PubMed Scopus Google Scholar). and a screening method for using the biomarker a of and of by tandem mass Chem. 2007; PubMed Scopus Google Scholar). were extracted in an with in min and for analysis. peptides were targeted to the of the using peptides from the as an internal This well with the of as by a well-established The and the of the method showed for population-wide screening. used a to newborns for associated with Newborn screening for using tandem mass Chem. PubMed Scopus Google Scholar). more than DBS samples by targeting tryptic peptides were to four and their with a method measured the proteins obtained from whole blood samples. MRM approach was to the for all targeted a method for screening newborn DBS samples for a protein to A. K. M. peptide of in dried blood spots using liquid mass application to newborn Chem. PubMed Scopus Google Scholar). method peptides with LC/MRM-MS and to those from an for the of samples. The monitoring of a protein in blood was with to the of this approach for preclinical D. M. of DBS for the of a protein using or 2011; 3: PubMed Scopus Google Scholar). Finally, a multiplexed approach was by for the quantitation of proteins in DBS samples collected from of proteins of dried blood spot samples and by methods in PubMed Scopus (41) Google Scholar). In all only to proteins were targeted in DBS samples and the of MRM were not methods using peptides have at highly multiplexed quantitation of proteins in plasma and serum samples mass multiple reaction monitoring for plasma Full Text Full Text PDF PubMed Scopus Google Scholar, T.A. S. T. A. M. T.A. D. D. A. S. M. P. of the and of multiple reaction measurements of proteins in 2009; PubMed Scopus Google Scholar, D. of in PubMed Scopus Google Scholar). work the for integrating DBS sampling with LC/MRM-MS for highly multiplexed quantitation of endogenous proteins. Many of the 60 proteins we have targeted have been or by and are at a in clinical The assay in this a highly reproducible method for multiple proteins from DBS samples by and with their were by a LC/MRM-MS been to and of proteotypic peptides in human plasma D. of in PubMed Scopus Google Scholar, D. of and liquid chromatography for quantitation of plasma biomarker Chem. PubMed Scopus Google Scholar). The from whole blood and the DBS samples were and the of DBS samples stored under been The panel was of 60 blood and of the as in in of proteins are or analytes in human plasma or and for their plasma concentrations are in clinical as The clinical plasma a of clinical for proteins in plasma and Chem. 2010; PubMed Scopus Google Scholar). have procedure for peptides for targeted proteins in M.A. D. reaction quantitation of proteins in human 2009; Full Text Full Text PDF PubMed Scopus Google Scholar, M.A. of MRM for plasma Meth. Scholar). and the are used to tryptic peptides are for protein within the human These peptides are on the of their in of for peptide and of and have used many of peptide targets to proteins in plasma samples D. of in PubMed Scopus Google Scholar, M.A. D. reaction quantitation of proteins in human 2009; Full Text Full Text PDF PubMed Scopus Google Scholar). In DBS 60 proteins were by 65 peptides with peptides for and and peptides for The peptides were using as M.A. D. reaction quantitation of proteins in human 2009; Full Text Full Text PDF PubMed Scopus Google Scholar). peptides a of an or or or at the The peptide was to and by of were The concentration of peptide was then by and capillary the of the the most abundant MRM for peptide were by on the mass with the as D. of in PubMed Scopus Google Scholar). The MRM were to the peptides in the LC/MRM-MS and A sample of whole human blood from and was from a were not and intravenous blood was collected in The blood was and stored use within Blood spots of μl volume were made on filter paper using an μl, The DBS collection were the of the was not in with any to of blood through the filter spotting was in a and were to for 4 The dried were then stored in with a of desiccant and a part the DBS samples from the storage the was to was less than the of of and in a of their in of the 60 blood proteins targeted in this DBS method M.A. A of the of and on the of human plasma proteins by Res. 2010; PubMed Scopus Google Scholar). In this we the most conditions in an at 37 to also for protein from the DBS collection as Samples were prepared using the DBS by the on the DBS collection which was then to in a were and reduced in a of 25 and at 60 °C at for of the protein was then to a and with 10 and with 10 for min at 37 °C. was at a concentration of μl of the blood sample and the was to for at DBS samples for the were prepared with an peptides were in at the concentrations as the endogenous peptides from a LC/MRM-MS of a plasma The was by the of and by at for Samples were by using the procedure for and LC/MRM-MS samples were with in for a with to the blood Samples for calibration curves were by of whole blood with an spotting onto DBS collection cards. A of concentration a range of and DBS samples were in for at concentration the blood was the to an in the spot on the collection the DBS within the was for analysis. Samples from all concentration were with the of peptides sample were using an with a to °C. A was in and was in of 10 μl to of the whole blood The was and the was of linear the 25 were then on an mass using the capillary at °C, at °C, and and to screening the for MRM the for the and of the 65 targeted peptides were a of The monitoring of was within min a of 60 the to the and were 10 and The most abundant MRM for peptide were for by LC/MRM-MS by monitoring the peptide in the peptide in and the peptide in blood. The for multiple of the peptide for the conditions in the the of an In this targeted peptides were considered to have this screening the coefficient of of the abundances of multiple peptide was for at least was using and MRM the most abundant was used for quantitation the as to the of the and peptides. extracted were to and integration by the a concentration to be in the calibration the of the must have been using linear with a of for the DBS sampling is most in clinical to newborns for in by targeting small molecule analytes I. Marsden D. Newborn Screening.Crit. Rev. Clin. Lab. Sci. 2009; 46: 55-82Crossref PubMed Scopus (41) Google Scholar). Samples are prepared by spotting a small drop of blood onto the DBS collection and only a of the DBS is for using a the volume of the blood into the assay is by the of the and not require of the volume of blood is are blood and analytes the filter These the blood spot and are in the as to or not the of the analytical Dried blood spot sampling in with for of small 2010; PubMed Scopus Google Scholar, Use of filter paper for the collection and of human whole blood PubMed Google Scholar). In newborn screening is made for and the associated with the use of DBS samples is P. in newborn 2011; 44: PubMed Scopus Google Scholar). applications small molecule have can be controlled to a <15% Dried blood spot sampling in with for of small 2010; PubMed Scopus Google Scholar). In of and quantifying multiple proteins from DBS samples, we to the of must be controlled in the sample by spotting of blood and the blood spot for analysis. also the DBS collection the and the of this and been through of use in many newborn screening The of dried blood spot samples using liquid chromatography tandem mass 2011; 44: PubMed Scopus Google Scholar). 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This excellent is in with of for multiplexed the for samples was less than D. of in PubMed Scopus Google Scholar). The linear dynamic range was by of the whole blood spotting on a DBS collection and in a of peptides protein DBS samples for concentration were prepared to for any variation in blood spotting and protein from the DBS collection at the concentration These samples were in the of concentration with sample the calibration for using the peptide was linear the range with an R2 value of Furthermore, the was <15% for the DBS samples prepared and for concentration This linear the protein and the protein for the targeted peptide is enabling protein Overall, a linear was for 40 of the peptides with an R2 value of The was than for peptides which targets for a clinical MRM many biomarker proteins by less than when and be to the linear dynamic range for of targeted proteins by to the sample protein concentration in the DBS samples was using the from peptide calibration the peptide and the protein molecule the 4 the 37 proteins quantified in DBS samples in of The concentration range of the proteins in this assay more than four orders of magnitude with at and at proteins by multiple peptides and the protein concentration in 4 was using the most abundant which to a of the protein was the most was by a protein concentration and the measured concentration of over using peptides and is the for a sample is not for all proteins M.A. A of the of and on the of human plasma proteins by Res. 2010; PubMed Scopus Google Scholar, of on the of proteins from Res. 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M. peptide of in dried blood spots using liquid mass application to newborn Chem. PubMed Scopus Google Scholar). we a tryptic peptide in LC/MRM-MS concentration of was to the concentrations in the method and the must the a single sample from 10 was used to the analytical of assay and the of measured protein concentrations to is only considered to be an the value of a clinical assay on to and patients and this be by quantitation of the endogenous to the concentration of the internal for the of the is not as important as and have can be multiple clinical samples in of plasma samples by MRM D. of in PubMed Scopus Google Scholar). Furthermore, have quantitation well with for proteins under G. P. T. can the protein in Chem. PubMed Scopus Google Scholar). In this we have this DBS assay the first as all quantified peptides were measured with high be required to protein on a of samples clinical value can be samples of whole blood were in with all of the DBS with the only the μl of the blood sample was not on the DBS collection to the protein protein an of the sample was centrifuged at for 10 min and the of the blood the of temperature, and for was to MRM were for using the procedure to DBS samples MRM the peptides in the were also in the DBS samples and the MRM for peptide were the for sample This the of the filter paper not to the sample and were to the DBS and were only in the of prepared samples in an of for the peptide which was to the for the peptide targets in the DBS samples. This the in spotting the sample onto DBS collection and the proteins not the variation in the Linear calibration curves were obtained for peptides in the whole blood samples, which well with the 40 peptides a linear in the DBS samples. A of 33 proteins were quantified in DBS and samples and the measured protein concentration in samples well with a slope of 0.96 and an R2 value of 0.97. This highly protein from the DBS collection was the protein concentration in DBS and samples have the of the DBS samples. in the sample have targeted peptides the samples DBS collection were used in DBS are to endogenous we have not their with in of and is important to storage conditions for DBS samples to the of the measured In approach to quantifying we are in the of targeted peptides as for the proteins and not the proteins themselves. peptide targets as which to sample and the conditions for DBS samples without on M. into the conditions ambient sample impact to dried blood spot sample 2011; 3: PubMed Scopus Google Scholar). work samples °C and °C and method −20 °C to 40 °C. the DBS samples stored at −20 °C, 4 °C and 37 °C for and 10 which are required to samples to a A blood sample was used in this a DBS sample was prepared and all MRM were to the screening for as for the DBS the 40 peptides quantified in the DBS were peptides also in DBS samples and the most abundant MRM used for was the for in this sample most the to tryptic from and from DBS samples were at the and were the storage −20 °C, 4 °C, and 37 °C. DBS samples from were and 10 days of the DBS samples were with peptides any in the protein or of the peptide storage be in the concentration The was by the concentration from the from or to the obtained on were considered stable their concentration storage was within of their measured peptides were in DBS samples storage for and 10 days at all The is in and is in the in days of storage only peptides were the The concentration of by and storage at −20 °C, 4 °C, and 37 °C. by at 37 °C storage and by at 4 °C in the protein concentration be by protein to tryptic for the the obtained 10 days of storage were to those A peptides are also at concentrations less than 80% of the the concentration of by and when stored at 4 °C and 37 °C. The concentrations be by reduced protein from the DBS collection or of the all was at days of was within the targeted range 10 days of The be of protein and storage a in by of the peptide at This the of the multiple the targeted protein and peptide of multiple peptides protein to the in is at the protein or peptide In was targeted by peptides and 10 days of storage at 4 °C, was to by by the in is and not to the of from the DBS collection Overall, sample was storage for and 10 and was of storage temperature −20 °C and 37 °C. are to the of the targeted peptides by the DBS samples or of the DBS be for the of The multiplexed quantitation of DBS samples by methods is an for population-wide screening for small and is on of method we have the first of DBS sampling for highly-multiplexed protein quantitation of endogenous proteins. The LC/MRM-MS approach and the to expand the was highly reproducible in high measurements of protein concentration those from traditional plasma or serum samples. The majority of the targeted proteins were to be quantified in DBS samples and excellent over days of at These quantitation of endogenous proteins in DBS many in evaluating the of this technology for clinical The of in the blood spotting spotting and on the and be for protein in a clinical environment. In addition, larger sampling and the of samples with methods for of the of measured protein the of this for protein quantitation in DBS samples and the required for clinical

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