Dataset Information

Multiplexed MRM concentration measurements of >200 endogenous proteins in dried blood spots for biomarker discovery and validation

ABSTRACT: The use of protein biomarkers as surrogates for clinical endpoints requires extensive multilevel validation including development of robust and sensitive assays for precise measurement of protein concentration. Multiple reaction monitoring (MRM), is a well-established methodology that can be used for reproducible protein-concentration measurements in biological specimens collected via microsampling. The dried blood spot (DBS) microsampling technique can be performed non-invasively without the expertise of a phlebotomist, and can enhance analyte stability which facilitate the application of this technique in retrospective studies. Thus, precise, sensitive, and multiplexed methodologies for measuring protein concentrations in DBSs can be used for de novo biomarker discovery. To achieve this goal, MRM assays were developed for multiplexed concentration measurement of proteins in DBS. The lower limit of quantification (LLOQ) was found to have a median total coefficient of variation (CV) of 18% for 245 proteins, while the median LLOQ was 5 fmol of peptide injected on column, and the median inter-day CV over 4 days for measuring endogenous protein concentration was 8%. The majority (88%) of the assays displayed parallelism, while the peptide standards remained stable throughout the assay workflow and after exposure to multiple freeze-thaw cycles. For 190 proteins, the measured protein concentrations remained stable in DBS stored at ambient laboratory temperature for up to 2 months. Finally, the developed assays were used to measure the concentration ranges for 200 proteins in twenty same sex, same race and age matched individuals.

ORGANISM(S): Homo Sapiens

SUBMITTER: Azad Eshghi

PROVIDER: PXD015118 | panorama | Mon Jan 06 00:00:00 GMT 2020

REPOSITORIES: PanoramaPublic

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Publications

Concentration Determination of >200 Proteins in Dried Blood Spots for Biomarker Discovery and Validation.

Eshghi Azad A Pistawka Adam J AJ Liu Jun J Chen Michael M Sinclair Nicholas J T NJT Hardie Darryl B DB Elliott Monica M Chen Lei L Newman Rachael R Mohammed Yassene Y Borchers Christoph H CH

Molecular & cellular proteomics : MCP 20200102 3

The use of protein biomarkers as surrogates for clinical endpoints requires extensive multilevel validation including development of robust and sensitive assays for precise measurement of protein concentration. Multiple reaction monitoring (MRM) is a well-established mass-spectrometric method that can be used for reproducible protein-concentration measurements in biological specimens collected via microsampling. The dried blood spot (DBS) microsampling technique can be performed non-invasively w ...[more]

PMID: 31896676

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Project description:We examine the use of high-throughput sequencing on binding sites recovered using a bacterial one-hybrid (B1H) system and find that improved models of transcription factor (TF) binding specificity can be obtained compared to standard methods of sequencing a small subset of the selected clones. We can obtain even more accurate binding models using a modified version of B1H selection method with constrained variation (CV-B1H). However, achieving these improved models using CV-B1H data required the development of a new method of analysis - GRaMS (Growth Rate Modeling of Specificity) - that estimates bacterial growth rates as a function of the quality of the recognition sequence. We benchmark these different methods of motif discovery using Zif268, a well characterized C2H2 zinc finger transcription factor on both a 28bp randomized library for the standard B1H method and on 6bp randomized library for the CV-B1H method for which forty-five different experimental conditions were tested: 5 time points and three different IPTG and 3-AT concentrations. We find that GRaMS analysis is robust to the different experimental parameters whereas other analysis methods give widely varying results depending on the conditions of the experiment. Finally, we demonstrate that the CV-B1H assay can be performed in liquid media, which produces recognition models that are similar in quality to sequences recovered from selection on solid media. All selections were performed using the bait transcription factor Zif268. 45 different B1H selections were performed on plates and the selected sites were multiplexed and sequenced in a single lane. A single selection was performed in liquid media and sequenced along with other multiplexed sets of sites in a separate lane. For the experiments performed on plates, the of the selections assays was varied (4, 8, 12, 18, 24 hours). The stringency of the selections was also varied by changing the concentration of IPTG (0, 10, 50 uM) and 3-AT (0.5, 1, 2 mM). The initial randomized library used for all selections was also sequenced in a single lane. The randomized portion of each transcription factor binding site is 6bp long and is immediately 5' of the constant sequence, GCGG, which is the consensus binding site for finger 1 of Zif268. Upstream of the randomized binding site is a 4bp randomized region referred to as the key region. The purpose of the key region is to detect possible PCR 'jackpotting.' The single liquid media experiment was run for 4 hours using 50uM IPTG and 5mM 3-AT. Four selections were performed using a prey library with a 28bp long randomized region and not fixed consensus site region. These selections were performed at either 36 or 48 hours using 10uM IPTG and 0, 2, or 5 mM 3-AT.

Dataset Information

Multiplexed MRM concentration measurements of >200 endogenous proteins in dried blood spots for biomarker discovery and validation

Publications

Concentration Determination of >200 Proteins in Dried Blood Spots for Biomarker Discovery and Validation.

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