Authorship:
Andrew G. Chambers, Andrew J. Percy, Juncong Yang, and Christoph H. Borchers
UVic-Genome BC Proteomics Centre, Victoria, BC
| Short Abstract We report a multiplexed LC-MRM-MS method for the precise quantification of 97 endogenous proteins in human DBS samples, suitable for biomedical research applications. Standard curves were generated for each of the 173 target peptides (representing the 97 proteins) to fully characterize the assay’s analytical merits. Furthermore, the stability of each target peptide was investigated in DBS samples stored at -20°C, 4°C, RT and 37°C over a duration of 154 days. Finally, we will present a simple strategy for managing the hematocrit effect across samples and discuss the implications on the DBS-MRM workflow. |
Long Abstract
Introduction:
The integration of dried blood spot (DBS) sample collection with mass spectrometry (MS) has proven advantageous for quantifying small molecules in biomarker screening and drug development. To leverage these benefits for growing proteomic applications, we report a multiplexed LC-MRM-MS method for the precise quantification of 97 endogenous proteins in human DBS samples.
Methods:
All blood used in method development was collected from healthy, non-fasting individuals. Both individual and pooled whole blood samples were spotted on DBS collection cards (Whatman 903), dried under ambient conditions, and stored under desiccant for various durations. DBS samples were then transferred to a 96-well plate for extraction, denaturation, and digestion with trypsin. Matching stable isotope-labeled standard (SIS) peptides were spiked in the digests to enable precise, relative quantitation of targeted proteins. All liquid handling steps in sample preparation were automated on a Tecan Freedom EVO 150 robotic system. Samples were then analyzed by UHPLC-MRM/MS on an Agilent 6490 triple quadruple instrument. The MRM data was processed using a combination of Agilent MassHunter and in-house developed software for automated standard curve generation and qualification.
Results:
Standard curves were generated for each of the 173 target peptides (representing the 97 proteins) to fully characterize the multiplexed MRM assay. The linearity was excellent with an average linear dynamic range was 207-fold and an average R2 value of 0.986. The endogenous concentration of these quantified proteins spanned almost 5 orders of magnitude from serum albumin at 18.0 mg/mL down to cholinesterase at 190 ng/mL. The average CV for the intra-assay precision (repeatability) and inter-assay precision (reproducibility) ranges for 6 biological samples were 6.1-7.5% and 9.5-11.0%, respectively. Furthermore, the stability of each target peptide was investigated in DBS samples stored at -20°C, 4°C, RT and 37°C over a duration of 154 days. The percentage of stable peptide targets was 95-98% after 2 days, 72-76% after 29 days, and 52-66% after 154 days. Interestingly, peptide stability was largely independent of the storage temperature. Finally, we will present a simple strategy for managing the hematocrit effect across samples and discuss the implications on DBS sample preparation.
Conclusions:
We have developed a robust MRM method for quantifying 97 proteins in human DBS samples, representing the highest multiplexing reported for DBS-MRM proteomics. All liquid handling steps in the sample preparation have been automated on the 96-well format and results are both highly repeatable and reproducible. This multiplexed MRM assay has been validated for biomedical research applications, such as supporting biomarker verification and validation.