Dynamic and temporal assessment of human dried blood spot MS/MSALL shotgun lipidomics analysis
© The Author(s). 2017
Received: 20 December 2016
Accepted: 13 March 2017
Published: 20 March 2017
Real-time and dynamic assessment of an individual’s lipid homeostatic state in blood is complicated due to the need to collect samples in a clinical environment. In the context of precision medicine and population health, tools that facilitate sample collection and empower the individual to participate in the process are necessary to complement advanced bioanalytical analysis. The dried blood spot (DBS) methodology via finger prick or heel prick is a minimally invasive sample collection method that allows the relative ease and low cost of sample collection as well as transport. However, it has yet to be integrated into broad scale personalized lipidomic analysis. Therefore, in this study we report the development of a novel DBS high resolution MS/MSALL lipidomics workflow.
In this report we compared lipidomic analysis of four types of blood sample collection methods (DBS, venous whole blood, serum, and plasma) across several parameters, which include lipidomics coverage of each matrix and the effects of temperature and time on the coverage and stability of different lipid classes and molecular species. The novel DBS-MS/MSALL lipidomics platform developed in this report was then applied to examine postprandial effects on the blood lipidome and further to explore the temporal fluctuation of the lipidome across hours and days.
More than 1,200 lipid molecular species from a single DBS sample were identified and quantified. The lipidomics profile of the DBS samples is comparable to whole blood matrix. DBS-MS/MSALL lipidomic analysis in postprandial experiments revealed significant alterations in triacylglyceride species. Temporal analysis of the lipidome at various times in the day and across days identified several lipid species that fluctuate as a function of time, and a subset of lipid species were identified to be significantly altered across hours within a day and within successive days of the week.
A novel DBS-MS/MSALL lipidomics method has been established for human blood. The feasibility and application of this method demonstrate the potential utility for lipidomics analysis in both healthy and diverse diseases states. This DBS MS-based lipidomics analysis represents a formidable approach for empowering patients and individuals in the era of precision medicine to uncover novel biomarkers and to monitor lipid homeostasis.
KeywordsHuman Dried blood spot Shotgun lipidomics Mass spectrometry
For over two decades the dried blood spot (DBS) methodology has commonly been used in the diagnosis of inborn errors of metabolism [1–3]. Currently, more than 95% of newborns are screened for inherited metabolic disorders using DBS in the United States [2, 4]. Using this methodology blood samples are typically obtained from heel or finger pricks and spotted onto filter paper for collection and analysis. DBS offers several advantages over conventional whole blood, plasma or serum sample collection [5, 6]. For example, DBS sample collection is minimally invasive and easy to perform (e.g. finger or heel prick rather than venous puncture) and notably, it can be done at home by patients or volunteers themselves after minimal training. In addition, a low volume of blood (less than 20 μL) is needed to spot onto filter paper compared to a minimum of 0.5 mL of blood in venous sampling. Lastly, DBS is relatively stable at ambient temperature, thus facilitating easy shipping and storage of samples. As such, the application of DBS has been developed for therapeutic drug monitoring [7, 8], pharmacokinetics , genomics , proteomics [11, 12], and metabolomics .
The lipidome of human blood is comprised of thousands of lipid molecular species . Its homeostatic regulation plays a pivotal role in numerous pathological disease states, such as atherosclerosis, cancer, metabolic diseases, and Alzheimer’s disease [15–20]. Lipidomic analysis is largely based on electrospray ionization mass spectrometry (ESI-MS) coupled with liquid chromatography (LC) or using direct infusion, i.e., shotgun lipidomics [21–23]. Using the ESI-MS/MS strategy, lipid molecular species can be identified by their characteristic fragments (from the head group or fatty acid chains) and their molecular weights. Typically, these types of ions have been applied in precursor ion scanning, neutral loss scanning, and multiple reaction monitoring experiments to measure lipid species from human serum, plasma, and whole blood [23, 24]. Traditional MS-based lipidomics has been dominated by triple quadrupole instruments and require the pre-selection of specific ions or multiple runs for the diverse capture of the lipidome. Recently, a novel data-independent acquisition (DIA) technique of mass spectrometry has been developed to overcome this shortcoming by parallelizing the fragmentation of all detectable ions within a certain m/z range [25, 26]. With the combination of high-resolution MS, DIA provides a powerful tool for shotgun lipidomics. MS/MSALL is a direct infusion DIA technique, using a hybrid quadrupole time-of-flight (QTOF) technology, specifically designed for lipidomics [27, 28]. In MS/MSALL, all precursors are selected in the Q1 quadrupole at unit-based resolution in a step-wise fashion to completely cover the entire mass range of interest. Collision-induced dissociation is carried out in Q2 at high speed and the corresponding high-resolution MS/MS spectra are recorded covering every precursor in each cycle . The combination of high resolution, sensitivity, and throughput offers significant advantages towards the identification and quantification of lipids in complex biological extracts.
Here in, we report a high-throughput DBS-MS/MSALL lipidomics platform to analyze the blood lipidome via the direct infusion of lipids extracted from DBS samples. More than 1,200 lipid molecular species were identified and quantified in a single dried blood spot sample. We also explored the lipidomics profiles from four different blood matrixes: 1) dried blood spot from a finger prick, 2) whole blood (through venous sampling), 3) plasma, and 4) serum. In addition, DBS lipid stability at various storage temperatures and time points was assessed. The high-throughput DBS-MS/MSALL shotgun lipidomics workflow demonstrated here exemplifies a robust and formidable tool for investigation of population health and precision medicine in diverse disease states and interventions that empower individual participation and avoids unnecessary sample waste.
All lipid standards were purchased from Nu-Chek Prep Inc (Waterville, MN), Avanti Polar Lipids (Alabster, AL), Cayman Chemical (Ann Arbor, MI), Matreya (State College, PA), Cambridge Isotope Laboratories (Tewksbury, MA), or Sigma-Aldrich (St Louis, MO). All solvents were of HPLC or LC/MS grade and were purchased from Fisher Scientific (Waltham, MA) or VWR International (Radnor, PA). Whatman 903™ filter paper cards, adjusted ACCU-CHEK lancets, and Uni-Core punch with ID 6 mm were purchased separately from GE Healthcare (Westborough, MA) and Roche Diagnostics (Indianapolis, IN).
Preparation of dried blood spots
Written informed consent was provided by the subjects prior to participating in the study. Research use of samples was conducted in accordance with the terms outlined within the informed consent form and the terms set forth therein and with the tenets of the Declaration of Helsinki. Diets and other physiologic parameters were not controlled for in this study.
For the blood matrix study, the volunteer was fasted overnight (around 12 h) and venipuncture of a cubital vein was performed in the morning. Blood was drawn into a non-coated tube for whole blood, a K2 EDTA tube (BD, Franklin Lakes, NJ) for blood plasma, and silicone coated tube (BD) for blood serum. For whole blood samples, tubes were mixed well by gentle inversion 10 times and whole blood was immediately spotted on Whatman 903™ filter paper cards. For serum and plasma samples, the tubes were centrifuged at 1,200 g for 15 min at room temperature and the supernatant plasma and serum were spotted onto Whatman 903™ filter paper cards. Concomitantly, blood via finger prick was taken and spotted onto Whatman 903™ filter paper card. All samples were air dried for 3 h at room temperature (~22 °C) and collected using Uni-Core punch (ID 6 mm, GE Healthcare) for extraction as described below.
For the DBS stability study, whole blood was drawn and immediately spotted onto Whatman 903™ filter paper cards. After samples were air dried completely for 3 h at room temperature, the paper cards were stored with desiccant at different conditions: 4 °C, room temperature, and 37 °C for 3 days, 1 week, and 2 weeks.
For the time course study, a total of 12 healthy volunteers (6 male and 6 female) were included (Mean age 31 ± 6 years). After finger pricks using a single-use safety lancet, the blood drop was directly applied onto Whatman 903™ filter paper cards at 7 AM (after overnight fasting), 10 AM, 1 PM (1 h after lunch), 4 PM, and 7 PM. The blood spots were air dried completely for 3 h at room temperature and stored at room temperature in a zip-closure bag with desiccant.
For the DBS daily variation study, a total of 16 healthy volunteers (8 male and 8 female) were included (Mean age 32 ± 6 years). Finger prick blood samples were collected onto Whatman 903™ filter paper cards in the morning after overnight fasting over 5 consecutive days. The blood spots were air dried completely for 3 h at room temperature and stored at room temperature in a zip-closure bag with desiccant.
Sample preparation and extraction
The dried blood spots were punched out uniformly using a GE Healthcare Uni-Core punch with ID 6 mm and transferred to glass tubes. Lipid extraction was performed using a modified Bligh and Dyer as previously described [22, 27, 29]. Four mL chloroform:methanol (1:1, v/v) and 1.6 mL LiCl solution (50 mM) were added to each sample with a cocktail of deuterium-labeled, or odd chain and extremely low naturally abundant fatty acid internal standards for diverse lipid classes (Additional file 1: Table S1). The extraction homogenate was vortexed and centrifuged at 1000 g for 5 min. The chloroform layer of each extract mixture was carefully removed and saved. An additional 2 mL chloroform was added into the MeOH/aqueous layer of each test tube. After centrifugation, the chloroform layer from each individual sample was combined and dried under a nitrogen stream. Each individual residue was then re-suspended in 4 mL chloroform/methanol (1:1), back-extracted against 1.8 mL LiCl aqueous solution (10 mM), and the extract was dried as described above. Such lipid extraction was automated using a customized sequence on a Hamilton Robotics STARlet system (Hamilton, Reno, NV) designed to meet high-throughput requirements. Finally, lipid extracts were dried under nitrogen and reconstituted in 68 μL chloroform:methanol (1:1, v/v). Samples were flushed with nitrogen and stored at -20 °C. The reproducibility of the extraction method was evaluated by measuring the peak area of the added internal standard in three replicates of DBS. The recovery was obtained by comparing the response of analyte added and extracted from the DBS to the response for the analyte in solvent. The sensitivity was assessed as the limit of quantification (LOQ) for each added internal standard by measuring the serial dilution of the extracted DBS samples (Additional file 2: Table S2).
Direct Infusion Quadruple Time of Flight (QTOF) mass spectrometry
Summary of polarity and scan modes for lipid classes using ESI-MS/MSALL
MS/MS Scan Mode
M + H
Precursor ion of 184.1
M + H
Neutral loss of 141.0
M + H
Precursor ion of 184.1
M + NH4
Neutral loss of FAb
M + NH4
Neutral loss of FA
M + H
Precursor ion of 264.2
M + H
Precursor ion of 85.0
M + H
Precursor ion of 264.2
Precursor ion of FA
Precursor ion of FA
Precursor ion of FA
Precursor ion of FA
The results are expressed as mean ± standard deviation (SD). Statistical analysis comparing various blood matrices coverage was performed using Graphpad Prism. The analysis of lipids over time was conducted using a repeated one-way analysis of variance ANOVA using R programming . The principal component analysis (PCA) was carried out for the different blood matrix study using R programming .
Adaption of DBS-MS/MSALL shotgun lipidomics to different blood matrix samples
The choice of negative mode or positive mode analysis for different lipid classes is based on the differential propensity of each lipid class to acquire either positive or negative charges under the source of high voltage [15, 22, 33]. Anionic lipids, including PA, PG, PI, and PS are acquired under the negative modes for the higher sensitivity. The zwitterion phospholipids, such as PC, PE, and SM, and the neutral lipids, prefer to become either the protonated ions or ammonium added ions in the positive modes. For each different lipid class, the quantification was carried out by either precursor ion scanning or neutral loss scan, specific for acyl fatty acid anions, or lipid class-specific fragment ions [22, 24]. The ionization mode, molecular ions, and their corresponding scan modes are summarized in Table 1.
Lipid extraction is one of the key steps to the successful analysis of shotgun lipidomics by ESI/MS in general. In this study, the modified Bligh and Dyer method was used to extract total lipids from DBS samples and is suited for analyzed lipid classes [22, 33]. LiCl solution was used to improve the extraction efficiency of acidic lipids, prevent the degradation of plasmalogen molecular species, and decrease spectral complexity. At the final stage, an additional Bligh and Dye extraction against an aqueous phase with LiCl was used to further enrich the lipid extracts. To evaluate the efficiency of DBS lipid extraction, the repeatability and recovery of the extraction was investigated using the added internal standards. The recovery rates for most of added lipid analytes are in the range of 50 to 100% with satisfactorily reproducible measurements (coefficient of variation [CV] <10% for most added lipid analytes) (Additional file 2: Table S2). The sensitivity of the MSMSALL shotgun lipidomics, represented by LOQ, ranging from 0.10 to 1.62 nM, is consistent with the previous reports [22, 23, 27, 28, 33].
Short-term stability of DBS
Postprandial triglyceride profiling by DBS-MS/MSALL lipidomics
Temporal profiling of lipid species over time in a day using DBS-MS/MSALL shotgun lipidomics
Temporal changes of fasted samples collected over 5 days using DBS-MS/MSALL lipidomics
Dried blood spot (DBS) analysis is a convenient way to collect blood samples with several advantages over conventional blood collection methods. DBS has gained popularity in fields such as newborn screening, preclinical studies, and therapeutic drug monitoring [2, 4, 7, 8]. DBS coupled with LC-MS/MS system provides the capacity to analyze samples in a high throughput manner once coupled to robust analytical methods. Lipidomics analysis of whole blood, which is comprised of thousands of diverse lipid molecular species, is directly linked to an individual’s physiological, nutritional and health status [14, 35]. In this study, we combined DBS collection with high-resolution MS/MSALL shotgun lipidomics analysis to analyze the blood lipidome. We demonstrate in one DBS spot, several lipid classes and more than 1,200 lipid species were identified and quantified.
Direct infusion-based MS shotgun lipidomics provides comprehensive profiling and quantitation of lipid species from organic extracts of biological samples without the LC column separation . Conventionally, numerous tandem MS strategies have to be applied for different lipid classes, such as precursor ion scanning and neutral loss scanning, and it requires the pre-selection of target lipids and fragmentations. On the other hand, the high-resolution MS/MSALL method, which utilizes DIA analysis, does not require any pre-selection and records all information in high resolution MS/MS spectra, thus allowing for data interpretation following the analysis . It is noteworthy to mention that the MS/MSALL approach employs 1 m/z isolation window instead of the wider windows, because the wider isolation windows result in the disconnection between the fragment ions and precursors, complicating the analysis of the acquired data . Using the 1 Da isolation window not only allows us to collect all the fragments from the precursors, but maintains the relationship between the fragments and the precursor ions, which facilitates the identification of lipid molecular species through data processing. Collectively, the MS/MSAll approach is bias-free and delivers the high specificity with informative production ion spectra and high sensitivity with the limit of quantitation (LOQ) as low as the sub nM or nM range for most of lipids, and offers a solution for measuring the lipidomes in diverse clinical samples using a high throughput approach that enables application of lipidomics in broader population-based analysis.
The era of precision medicine is now taking center stage, accelerating the stratification of patients based on their molecular signatures . Genomics alone, however, will not be able to selectively define all patient populations prone to a disease or therapeutic response. Proteomic, metabolomic, and lipidomic workflows are emerging dynamic “omics” technology, which can be complementary to genomics. Thus, illuminating the complexity of environmental exposure and phenotype of health as well as disease states [37, 38].
In this study the postprandial TAG molecular species and the entire lipidome identified by the DBS-MS/MSALL lipidomics platform delivered important insight that enables the potential for personal lipidomics profiling to monitor physiological changes, which could be applied to the diagnosis and presence of disease. It is notable that there are higher variations of lipid molecular species within the day than within the week (across days) (Fig. 6a), which indicates that internal factors (such as circadian rhythms), and external factors (i.e. diet) have a profound effect on the blood lipid profiles. This is not surprising given that several lipid classes and species have been demonstrated to show diurnal variation in animal and human studies . Thus, these data implicate the need to consider time of day/circadian rhythm when assessing specific lipid species for comparisons across individuals. However, the consistency of the lipid profiles across days implies that fasted blood samples are more stable across days when assessed at the same time among various individuals. The lipid molecular signatures from the temporal changes of lipid species over different time periods (hours vs days) using DBS-MS/MSALL lipidomics demonstrates the feasibility to use this technique for biomarker discovery in population-based studies and highlights the innate temporal changes of the lipidome in diverse individuals. Taken together, the DBS-MS/MSALL lipidomics platform, with its simple blood collection and high throughput capacity could be applied for future large-scale population based lipidome profiling.
As a less invasive sampling method DBS offers a simple collection protocol, requires a significantly smaller blood volume than venous sampling, and is easy to store and transfer, which thereby reduces the infection risk to various pathogens [5, 6, 40]. DBS coupled with LC-MS/MS provides a highly sensitive and selective method for quantitative analysis of small molecules, lipids, peptides, and proteins [8, 11, 41]. The challenges for developing DBS-LC-MS/MS methods include the lack of extensive references for most molecules using DBS for blood sampling, the discrepancy among blood and plasma/serum, and the possible interaction of blood and/or analytes with the matrix of a DBS card [40, 42], which were addressed by some of the experiments reported in this manuscript.
In summary, here in we developed a novel DBS-MS/MSALL shotgun lipidomics workflow to profile the human blood lipidome. In a single DBS spot, multiple lipid classes and more than 1,200 lipid species were identified and quantified. The application of this method to the postprandial lipidomics profiles and the temporal changes of lipids over time demonstrated the potential for use in preclinical and clinical studies, as well as for population-based health studies. Analysis of an individual’s lipidome using this novel platform provides a valuable approach for monitoring health status in real-time and provides a comprehensive profile of a person’s lipid homeostatic state.
Analysis of variance
Atmospheric-pressure chemical ionization
Coefficient of variation
Dried blood spot
Electrospray ionization mass spectrometry
High performance liquid chromatography
Liquid chromatography/mass spectrometry
We thank Khampaseuth Thapa for editing the manuscript.
Financial support for all studies in this report was provided by BERG, LLC.
Availability of data and materials
All data generated or analyzed during this study are included in this published article.
FG, VT, and MAK conceived, designed, and directed the research. FG and MAK co-wrote the paper. FG, JM, EYC, HR, and JD carried out experiments, analyzed data. VKV, RS, and NRN oversaw the experiments. All authors read and approved the final manuscript.
All authors are current employees of BERG, LLC.
Consent for publication
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All studies on human subjects were conducted with ethical approval and consent to participate.
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