Rapid method towards proteomic analysis of dried blood spots by MALDI mass spectrometry

Clinical Mass Spectrometry 12 (2019) 30–36

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Clinical Mass Spectrometry journal homepage: www.elsevier.com/locate/clinms

Rapid method towards proteomic analysis of dried blood spots by MALDI mass spectrometry Grace M. Samenuk a,⇑, Andrea R. Kelley a, George Perry b, Stephan B.H. Bach a a b

Department of Chemistry, University of Texas at San Antonio, One UTSA Circle, San Antonio, TX 78249, United States Department of Biology, University of Texas at San Antonio, One UTSA Circle, San Antonio, TX 78249, United States

a r t i c l e

i n f o

Article history: Received 3 August 2018 Received in revised form 13 March 2019 Accepted 13 March 2019 Available online 15 March 2019 Keywords: MALDI Mass spectrometry Dried blood spots DBS Neonatal screening

a b s t r a c t Neonatal dried blood spots (DBS) are routinely utilized in the clinical setting as a diagnostic tool for various genetic disorders and infectious diseases. DBS allow for minimally invasive, small volume blood collection and are stored at room temperature. Neonatal whole blood and serum samples can be important in determining genetic risk factors and predicting infantile disease; however, at the present time, limited methods exist for rapidly analyzing DBS samples for their proteomic profile, years after samples have been collected. A novel method is presented for the extraction and analysis of target proteins and peptides from neonatal DBS using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Extraction parameters were optimized to achieve ideal signal intensity and resolution to obtain protein identifications. Samples were extracted from filter paper with 0.1% TFA in H2O for 72 h. The extract was subjected to enzymatic digestion, spotted on an ITO-coated glass slide, and washed in order to remove salts. Analysis of extracted blood spots from ten newborns was completed. Similarities and differences in the proteomic profile of the washed extracts are presented, herein, to verify the viability of this method for analysis of dated DBS samples. This method allows for analysis of DBS samples years after collection and can be utilized to correlate diseases or disorders manifesting later in life with potential risk factors presenting in the proteomic profile of the DBS collected at time of birth. Ó 2019 Published by Elsevier B.V. on behalf of The Association for Mass Spectrometry: Applications to the Clinical Lab (MSACL).

1. Introduction Dried blood spots (DBS) are routinely collected from newborns in order to screen for potential disorders (metabolic [1], thyroid [2], etc.) and infectious diseases [3]. DBS collection was first presented in 1963 as a novel approach to the detection of phenylketonuria (PKU), an inherited disorder that causes a build-up of phenylalanine in the body, in infants [4]. Today, by utilizing DBS analysis, many diseases and disorders can be detected shortly after birth using a number of biomarkers or mutations present in blood

Abbreviations: CHCA, a-cyano-4-hydroxycinnamic acid; DBS, dried blood spots; EtOH, ethanol; HPLC, high performance liquid chromatography; ITO, indium-tinoxide; LC–MS/MS, liquid chromatography tandem mass spectrometry; m/z, massto-charge; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; MS, mass spectrometry; NBS, newborn screening; PKU, phenylketonuria; SA, sinapinic acid; SLE, solid-liquid extraction; TFA, trifluoroacetic acid. ⇑ Corresponding author. E-mail addresses: [email protected] (G.M. Samenuk), andrea.kelley@utsa. edu (A.R. Kelley), [email protected] (G. Perry), [email protected] (S.B.H. Bach).

[5]. When not screened for early, these conditions can often go unnoticed, until severe and sometimes irreversible symptoms present. Research focused on the discovery of new disease indicators is ongoing and, after validation, these indicators are included in Newborn Screening (NBS) programs, which aim for early disease detection and targeted treatment [6]. A single DBS can be utilized to test for a variety of different diseases and disorders that may not be visible otherwise [7]. Included in current NBS programs are benchmark blood levels for the disease indicators associated with a number of disorders, such as cystic fibrosis [8,9] and congenital hypothyroidism [2,10]. DBS collection is simple and virtually painless. It involves either a finger or heel-prick and deposition of a certain volume of blood onto cellulose-based filter paper [11]. Samples can then be shipped and stored at ambient temperature, in contrast to plasma or whole-blood samples that require additional storage considerations [12]. Certain proteins and other potential biomarkers in DBS samples can remain stable for years, with no significant degradation reported [12].

https://doi.org/10.1016/j.clinms.2019.03.002 2376-9998/Ó 2019 Published by Elsevier B.V. on behalf of The Association for Mass Spectrometry: Applications to the Clinical Lab (MSACL).

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A primary means of DBS analysis involves protein-based immunoassays and antibody kits [11]. While these methods are crucial to targeted analysis, antibodies can often be nonspecific and yield false positives or be of insufficient sensitivity, which can mean additional time between sample collection and accurate diagnosis/treatment [13]. Additionally, since blood is often collected on filter paper attached to a pre-prepared screening card, poor sample collection (uneven distribution, insufficient volume, etc.) can be problematic [14]. This makes extraction of the blood sample and analysis by mass spectrometry (MS) attractive. Volume required for analysis is in the microliter range and the distribution or homogeneity of the sample on the spot is trivial. MS offers the ability to analyze a large number of samples, while maintaining selectivity and sensitivity. This robust, multi-analyte analysis is attractive for NBS programs (targeted analysis), as well as exploratory research into how certain proteins, mutations and deficiencies correlate to disease states. Previous multi-analyte mass spectrometric studies on DBS extracts have been reported using liquid-chromatography tandem mass spectrometry (LC–MS/MS) [7,15]. This high-throughput approach results in robust, selective data, but is not an ideal approach for a shotgun proteomic analysis. Complex solutions are separated before ‘‘parent” ions of interest (with a particular mass-to-charge ratio (m/z)) are selected and fragmented into ‘‘daughter” ions utilized for identification. However, analytes must be ionized in solution prior to introduction to the mass spectrometer, and multiple charge states of the same analyte can be present in solution. Therefore, the potential for multiple m/z signals corresponding to the same analyte must be accounted for, which may limit the ability to obtain quantitative data. This signal-splitting will result in poor sensitivity and multiple fragmentation pathways [16]. Additionally, signals corresponding to singly-charged species may not be observed in MS/MS analyses designed to detect a limited m/z range. An alternative mass spectrometric approach, utilized in the experiments described herein, is matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). This mode of analysis addresses the complications associated with LC-MS/MS analysis. Analytes are not required to be in solution and are instead co-crystallized with a matrix material that facilitates ionization of predominantly singlycharged species. This makes protein identification by MS/MS straightforward. Complex samples, including proteins, are routinely analyzed by MALDI-TOF MS without the need for prior separation [17–21]. Mass spectrometric methods previously applied to the analysis of DBS have utilized LC-MS/MS [22,23]. Many of the common issues observed in LC-MS/MS analysis for DBS are eliminated with the use of MALDI-TOF MS, except for the fact that less abundant ions can be suppressed by more abundant ions. This is commonly observed in most shotgun mass spectrometry approaches without tedious separation steps [24]. Our goal was to develop a rapid and robust method for analyzing dated neonatal DBS samples and obtaining qualitative proteomic information from the extracts. Due to the age of the samples, the extraction method is meant to maximize the amount of analyte recovered from the DBS. This method is useful for correlative research into potential neonatal markers for diseases manifesting later in an individual’s life. In the current study, MALDITOF MS, and the inherent MS/MS (LIFT) capabilities associated with the instrument utilized, allowed for the identification of over 25 proteins based on their respective peptide fragments following enzymatic digestion of extracts from DBS samples obtained from a double-blind study. We discuss the ability to retrieve these identifications from dated DBS and comment on the similarities and differences between the samples as it pertains to future, more targeted studies.

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2. Results and discussion In the present study, 10 neonatal DBS samples were analyzed by MALDI-TOF MS. DBS samples on filter paper were stored at ambient temperature for two years before analysis. No special storage conditions were reported for the received samples. Several solidliquid extraction (SLE) parameters were tested and optimized in order to facilitate the observation of the highest number of molecular species at a mass resolution and intensity sufficient for analyte identification. Different solvent systems were investigated for optimal extraction efficiency. Among those tested, 0.1% TFA in H2O yielded the detection of the largest range of proteins after extraction and subsequent washes. A time study was conducted in order to determine the optimal length of SLE (that which yielded the most abundant signals). The DBS samples were immersed in the extraction solvent and aliquots of the extract were tested at 24-hour intervals, for a total of 96 h. After optimization of the aforementioned parameters, each of the samples was prepared for MS analysis by MALDI-TOF. Fig. 1 is a representative mass spectrum, collected in linear positive mode, of intact proteins from an extracted DBS sample. It was not originally known how many molecular species, if any, were going to still be extractable from the dated DBS. Because intact proteins were the initial target analytes, the matrix material utilized was sinapinic acid (SA), and the m/z detection range was set to approximately 3000–20,000 Da. The extracts were subsequently subjected to enzymatic digestion by trypsin in order to create a peptide mass fingerprint and identify the unknown proteins. Upon enzymatic digestion of the extracts, the samples were spotted on ITO-conductive glass slides. Due to the amount of salts and background signal being observed in the mass spectra due to the nature of the extracted blood, the samples were subjected to a series of washes designed to remove salts and other potential adducts prior to analysis. A primary drawback to any MS analysis is signal splitting as a result of the addition of adducts (Na+, K+, etc.) to signals corresponding to analytes of interest. MALDI-TOF analysis is particularly prone to this, making identification of analytes difficult. To compensate, clean-up steps, or washes, are often employed prior to analysis to limit the amount of salts present in the sample. Here, a H2O wash is employed to remove the cationic salts, while a subsequent EtOH wash is used to remove excess H2O from the sample. It is important, during this process, that steps are taken to limit the potential for analyte loss. To ensure negligible loss, the amount of time the spotted ITO-slides are submerged in the washes is optimized per sample type. The wash solvents utilized were subsequently analyzed following the same protocols described herein to ensure no analyte transfer from sample to solvent was occurring. No significant transfer of peptide material to the wash solvent was found. Fig. 2 depicts two representative, mass spectra of digested peptides from the same extracted DBS sample. The two spectra are meant to highlight the importance and the necessity of the wash steps. A (top) represents a mass spectrum obtained from a digested DBS sample with the wash step omitted. The insert demonstrates ion suppression of the signal of interest (m/z = 1529.9, represented by [M+H]+) by cationic salt adducts (sodium (represented by [M +Na]+) and potassium (represented by [M+K]+)). The salts are endogenous to the sample medium. The most intense peak for this particular peptide is the [M+K]+ instead of [M+H]+, and multiple peaks result in signal splitting for the same peptide. In contrast, B (bottom) represents a mass spectrum obtained from a digested DBS sample after being subjected to the wash steps described. The most intense peak is representative of [M+H]+ and the cationic salt adducts are no longer significant. Additionally, the overall intensity is two-orders of magnitude higher in the spectra corre-

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Fig. 1. Representative linear positive MALDI-TOF mass spectrum of an undigested DBS extract sample. Sample was extracted with 0.1% TFA in H2O for 24 h.

sponding to the washed sample than in the spectra corresponding to the unwashed sample. The MALDI matrix utilized for all peptide analysis was a-cyano-4-hydroxycinnamic acid (CHCA), and the m/z detection range was 540–3500 Da. Identification of proteins in unwashed samples can be problematic due to an excess of salts which produce adducts. As shown in Fig. 2, peaks corresponding to the same analyte with the addition of multiple adducts can present themselves as independent peptide fragments. Inadvertent MS/MS performed on the peaks including salt adducts, instead of the primary [M+H]+ signal, can result in false identifications of proteins that are not actually present in the sample. The mass shift due to the adduct may not be taken into consideration during the database search; therefore, the result will be a misidentified protein. Additionally, signals corresponding to analyte-salt adducts can overlap with signals corresponding to separate [M+H]+ identities allowing for missed protein identifications. Table 1 is a compiled list of all the positive identifications stemming from the MS/MS analysis of the most intense/resolved peaks from the ten DBS samples. Significant peaks (signal-to-noise above 5) were fragmented into their structural components for each sample. Fragmentation spectra were collected by LIFT (the MS/MS technique inherent to the Bruker UltrafleXtreme mass spectrometer) and characterized using the MASCOT search database. The analytes identified in Table 1 were not necessarily found in all DBS samples. Table 2 outlines which identifications were confirmed in each sample (X). By visualizing the data in this manner, we can begin to understand which proteins are common in neonatal blood samples, and those that may be indicative of abnormalities. It is important to note that some common proteins found in blood, such as hemoglobin, are noticeably absent from Table 1. Since these samples were two years old, and stored in less than ideal conditions, it is assumed that certain proteins may have rapidly degraded. In a study by Adam et. al, the stabilities of two hemoglobin species from extracted DBS were investigated at different humidity levels. They found that even at low humidity, hemoglobin was significantly degraded in a short amount of time [25]. This problem could be circumvented, or delayed, with optimized storage conditions. Three proteins were identified to be present in all ten DBS samples. The identified proteins are constituents of heparan sulfate glucosamine 3-O-sulfotransferase 3B1 (m/z = 1316.5 Da), immunoglobulin heavy chain variable region, partial (m/ z = 1529.9 Da), and a modulator of apoptosis 1 (m/z = 1834.1 Da). Each of these proteins are widely expressed throughout the body and in blood, and are part of the normal human proteome, and thus

we can use their consistent identification as a validation tool for our methods. In contrast, there are many proteins that were identified as being present in a majority of the DBS samples, or unique to only one or two DBS samples. The differences could account for the variation between prenatal environments, or potential indicators of future disorders. The ability to distinguish these similarities and difference by the described sample preparation and analysis steps confirms the potential usefulness of the method for time-delayed analysis of DBS samples. 3. Conclusions Neonatal DBS screening is a proven, viable method for the detection of early disease biomarkers and disorders in infants. The rapid method we have optimized has shown that with proper extraction and clean-up washes, certain proteins and other molecular species can still be analyzed and identified even after two years of ambient temperature storage. This is important for exploratory research of previously unidentified proteins that may be related to disease progression in individuals. DBS cards can be stored for years with no special storage; however, improved storage conditions, such as optimizing temperature or humidity, could result in prolonging the degradation process observed in some labile molecular species. 4. Methods 4.1. Dried blood spot origin DBS samples were procured from Dr. Braulio Jimenez-Velez at the University of Puerto Rico. Ten neonatal DBS samples were analyzed using the following methods. 4.2. Reagents Sinapinic acid (SA) (CAS# 530-59-6) (Sigma Aldrich) a-Cyano-4-hydroxycinnamic acid (CHCA) (CAS# 28166-41-8) (Sigma Aldrich) Trypsin, MS grade (CAS# 9002-07-7) (Sigma Aldrich) HPLC-grade H2O (CAS# 7732-18-5) (Thermo Fischer) Ethanol (200 proof, CAS# 64-17-5) (Thermo Fischer) Trifluoroacetic acid (0.1% in H2O, CAS# 76-05-1) (Sigma Aldrich)

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Fig. 2. (A) Representative reflectron positive MALDI-TOF mass spectrum of a digested, unwashed DBS extract sample, and (B) representative reflectron positive MALDI-TOF mass spectrum of a digested, washed DBS extract sample. (Spectra were collected on same sample, and the intensity scales are similar).

4.3. Extraction optimization and sample preparation for mass spectrometry analysis One-fourth of the outlined spot of the DBS was cut from the card stock and immersed in 500 mL of 0.1% TFA in H2O. Samples were vortexed for 1 min and left in solvent for 72 h for optimal extraction. After 72 h, 5 mL of the solvent extract was incubated with 4 mL of 0.01 mg/mL trypsin overnight at 37 °C. 4 mL digested extracts were hand-spotted on an ITO-glass slide (Delta Technologies, Limited, Loveland, CO. Part# CB-50IN-5111) and dried under vacuum. The slide was subjected to a 45 s HPLC-grade H2O wash and a 45 s 100% EtOH wash. Approximately 50 mL of solvent was utilized for each wash. These washes are designed to limit adducts and ion suppression observed in the mass spectra due to salts and detergents native to the samples. The ITO-slides were dried again under vacuum before matrix application. Each digested spot was

hand-spotted with 4 mL saturated CHCA and dried for a final time under vacuum before MS analysis. The additional three-fourths of the outlined spots in select samples were used to optimize the above parameters (extraction solvent, time, and washes).

4.4. MALDI-TOF analysis Mass spectrometric analysis was done in reflectron, positive ion mode on a time-of-flight mass spectrometer (UltrafleXtreme; Bruker Daltonics, Bremen, Germany). For the tryptic peptide analysis, the following parameters were optimized to maximize signal intensity and mass resolution: pulsed ion extraction was set to 150 ns; laser rate was set at 500 shots at 500 Hz per spectra; the ion source was set at 25 kV; and the m/z at which spectra was collected was 540–3500 Da. For the intact protein analysis, the same

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Table 1 A complete list of the MS/MS identifications from the DBS samples made by LIFT and confirmed using the MASCOT database. * indicates the peptide is present in all ten of the DBS samples analyzed. [M+H]+

Identification

Sequence

NCBI or GenBank reference

608.2 650.1 855.4 877.4 974.5 1048.7 1084.7 1109.6 1126.7 1194.7 1274.9 1282.6 1306.8 1316.5* 1430.8 1484.5 1529.9* 1639.8 1694.1 1732.3 1834.1* 1856.0 1871.9 2006.2 2301.3 2322.5 2341.4 2375.3 2426.2 3140.4

sex determining region Y, partial unknown, partial ras-specific guanine nucleotide-releasing factor 1 isoform 1 vasculin isoform 1 OVO-like 1 binding protein unnamed protein product mucolipin 1 variant, partial T-cell receptor gamma chain, CDR3 region, partial proapoptotic nucleolar protein 1 FAM230A, partial U2 snRNP-associated SURP motif-containing protein isoform 3 immunoglobulin heavy chain variable region, partial immunoglobulin G heavy chain variable region, partial heparan sulfate glucosamine 3-O-sulfotransferase 3B1 JAKMIP2 protein, partial coiled-coil protein immunoglobulin heavy chain variable region, partial neuronal acetylcholine receptor subunit beta-4 isoform 2 precursor amyloid-beta A4 precursor protein-binding family A member 1 GS homeobox 1 modulator of apoptosis 1 N-acylsphingosine amidohydrolase (acid ceramidase) 1, isoform CRA_b immunoglobulin heavy chain variable region, partial immunoglobulin heavy chain, partial protein FAM171A2 precursor olfactory receptor 14A2 hCG1982192, isoform CRA_ad hCG2011737 BTB (POZ) domain containing 7, isoform CRA_e type II gonadotropin-releasing hormone receptor, partial

QLGYQ DEFHC HLIICTR GGYHGGSSR RHLCTGCGK ERTAECALR LVNVSIHFR ALWEVSNYK ARLPAPRSAST GNLCGCIQGDSK LLCYRHLKTK EEHLVESGGGLR KAIAYADSVRGR LSGGRSCLDVPGR AGDGSEHCSSPDLR RAGAVGYSRSPSCSS SLSLSCTASGLTFSR GPTLPSVTEGPLGCGVR LNIVRCPPVTTVLIR AGAMPRSFLVDSLVLR KVALVGLTAETSHALVPK GATGRGSRGGGGVACPRPSR VGAMALDYGMDVWGQGTT VSCKASGGSFSTYAISWVR LGGEAGAAGVGDEPAPPEGTAPGPAR ANVTLVTGFLLMGFSNIQKLR AGSGQLHYSIPEEAKHGTFVGR GIFQLDQPDGLTVHSHTIQLR AYALNCGEGATVSYEIQIRVLR HPLGSAAGEEVWAGSGVEVEGSELPTFSAAAK

ADG26743.1 AAX93208.1 NP_002882.3 NP_075064.1 AAB72084.1 BAB71444.1 BAD96393.1 CAA10793.1 NP_001280096.1 AFU55684.1 NP_001307149.1 ACF36859.1 AEX28487.1 NP_006032.1 AAH03189.1 BAA76711.1 ACE74998.1 NP_001243496.1 NP_001154.2 EAX08418.1 NP_071434.2 EAW63792.1 CEF93576.1 AAS85822.1 NP_940877.2 NP_001342221.1 EAW62015.1 EAX09082.1 EAW81529.1 AAL27000.1

Table 2 A comparison of the similarities and differences of the identifications found in the ten DBS samples. Identification

Sample Identifiers ST01 ST02 ST03

sex determining region Y, partial unknown, partial ras-specific guanine nucleotide-releasing factor 1 isoform 1 vasculin isoform 1 OVO-like 1 binding protein unnamed protein product mucolipin 1 variant, partial T-cell receptor gamma chain, CDR3 region, partial proapoptotic nucleolar protein 1 FAM230A, partial U2 snRNP-associated SURP motif-containing protein isoform 3 immunoglobulin heavy chain variable region, partial immunoglobulin G heavy chain variable region, partial heparan sulfate glucosamine 3-O-sulfotransferase 3B1 JAKMIP2 protein, partial coiled-coil protein immunoglobulin heavy chain variable region, partial neuronal acetylcholine receptor subunit beta-4 isoform 2 precursor amyloid-beta A4 precursor protein-binding family A member 1 GS homeobox 1 modulator of apoptosis 1 N-acylsphingosine amidohydrolase (acid ceramidase) 1, isoform CRA_b KIAA1627 protein, isoform CRA_b immunoglobulin heavy chain variable region, partial immunoglobulin heavy chain, partial protein FAM171A2 precursor olfactory receptor 14A2 hCG1982192, isoform CRA_ad hCG2011737 BTB (POZ) domain containing 7, isoform CRA_e type II gonadotropin-releasing hormone receptor, partial

X

ST04

X

ST06

X X X

X X X X

ST05

ST07

ST08

X X X

X X X X X

X X X

X

X

X

X X X X

X

X

X X

X

X

X X

X

ST10

X

X X

X X

X X

X X X X

X X X

X X X

X X

X

X

X X X

ST09

X X

X X X

X X X X X X X X X X X X X X

X X X

X X X

X X X

X X X

X X X

X X

X X

X X X

X X X

X X X

X

X X

X

X

X

X X

X X X

X X X X

X X

X X X X X

X X

X X

X X

X

X X X

X

X

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Fig. 3. Method validation spectrum showing the recovery of angiotensin 2–7. The peaks at 784.5 Da represent the protonated molecular ion. The two visible peaks represent 50 ppm and 5 ppm of angiotensin.

parameters mentioned above were used, with the exception of the m/z at which the spectra were collected, which was 3000 to 20,000 Da. 4.5. Method validation Bovine blood samples were spiked with angiotensin 2–7 (Thermo Fisher) at three given concentrations to additionally assess the validity of our method. This is a small peptide with a molecular weight of approximately 783 Da, which falls in the range of our analytes. The three concentrations of angiotensin 2–7 that were spiked into the digested blood were 0.5 ppm, 5 ppm, and 50 ppm. The slide was subsequently washed and prepared for analysis using the procedures described herein. MALDI-TOF analysis indicated a peak corresponding to the protonated angiotensin 2– 7 at the 5 ppm and 50 ppm levels. Fig. 3 shows the presence of angiotensin 2–7 in spiked blood samples. The concentrations listed can give a rough estimate of the limit of detection for angiotensin 2–7; however, no absolute quantification should be deduced from this validation, since we are looking for qualification data. Additionally, the reproducibility between patients was assessed. Two spots were provided per patient. Both spots from the same sample was prepared independent of each other and analyzed. No significant difference between the two spots was observed. Funding sources Financial support was provided by the National Institute on Minority Health and Health Disparities (G12MD007591) from the National Institutes of Health, the National Science Foundation under CHE-1126708, and the I-DISCOVER Program Grant no. 2014-38422-22078/project accession no. 1003637 from the USDA National Institute of Food and Agriculture. Work was also supported through the Semmes Foundation (Kelley and Perry). Conflict of interest The authors declare no competing financial interest. Acknowledgements The authors gratefully acknowledge Dr. Braulio Jimenez-Velez at the University of Puerto Rico, Medical Sciences Campus for the DBS samples.

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