TY - JOUR
T1 - Targeting Endogenous Adduction Level of Serum Albumin by Parallel Reaction Monitoring via Standard Additions and Intact Protein Measurement
T2 - Biological Dosimetry of Catechol Estrogens
AU - Huang, Yu Shan
AU - Lin, Yu Min
AU - Chen, Hsin
AU - Wu, Chih Hsing
AU - Syu, Chong Hua
AU - Huang, Ting En
AU - Do, Quynh Trang
AU - Chen, Shu Hui
N1 - Funding Information:
This work was supported by Ministry of Science and Technology (MOST), Taiwan, Republic of China (105-2113-M-006-014-MY3, 108-2113-M-006-007). S.H.C. would like to thank Dr. Albert Heck and his group at Utrecht University, Netherlands, for helpful discussions and hosting her summer leave supported by MOST (106-2918-I-006-010). The authors gratefully acknowledge Mr. Po-Jung Su for the use of Orbitrap MS belonging to the Instrument Center of NCKU.
PY - 2019/12/17
Y1 - 2019/12/17
N2 - Abundant blood proteins adducted by active electrophiles are excellent markers to predict the risk of electrophile-induced toxicity. However, detecting endogenously adducted proteins by bottom-up selective (or parallel) reaction monitoring (SRM/PRM) is challenging because of the high variability in sample preparation and detection as well as low adduction levels. Here, we reported a new approach in developing PRM methods by combining intact protein measurement with standard additions to target optimal conditions for detecting catechol estrogens (CEs)-adducted human serum albumin (HSA). Blood serum was added with multiple amounts of CEs to obtain serum standards. Intact protein measurement revealed two linear ranges of adduction levels (adducted-CE/HSA): 0.34-0.42 (R2 > 0.94) and 0.81-8.54 (R2 > 0.96) against the amount of added CEs, respectively. Six adduction sites were identified by trypsin (K20, C34, K73, K281, H338, K378) or chymotrypsin (K20, C34, K378) digestion. PRM methods targeting all adducted/nonadducted peptide pairs based on chymotrypsin or trypsin digestion were developed, and the data were compared with those obtained by intact protein measurement. Correlation plots indicated that chymotrypsin-PRM leads to poor sensitivity and largely underestimated protein adduction levels. Trypsin-PRM leads to sensitive and highly correlated (R2 > 0.91) protein adduction levels with a detection limit below the endogenous level and relative standard deviation <25%. As a proof of concept, clinical serum samples were examined by trypsin-PRM, and a slightly higher adduction level was observed for the obesity group when compared with the healthy group. This is the first report on determining adduction levels of blood proteins for long-term exposure to CEs. The standard addition approach can be generally applied to protein adductomics with resolvable mass increments by intact protein measurement to accelerate the development of bottom-up methods close to the inherent limit.
AB - Abundant blood proteins adducted by active electrophiles are excellent markers to predict the risk of electrophile-induced toxicity. However, detecting endogenously adducted proteins by bottom-up selective (or parallel) reaction monitoring (SRM/PRM) is challenging because of the high variability in sample preparation and detection as well as low adduction levels. Here, we reported a new approach in developing PRM methods by combining intact protein measurement with standard additions to target optimal conditions for detecting catechol estrogens (CEs)-adducted human serum albumin (HSA). Blood serum was added with multiple amounts of CEs to obtain serum standards. Intact protein measurement revealed two linear ranges of adduction levels (adducted-CE/HSA): 0.34-0.42 (R2 > 0.94) and 0.81-8.54 (R2 > 0.96) against the amount of added CEs, respectively. Six adduction sites were identified by trypsin (K20, C34, K73, K281, H338, K378) or chymotrypsin (K20, C34, K378) digestion. PRM methods targeting all adducted/nonadducted peptide pairs based on chymotrypsin or trypsin digestion were developed, and the data were compared with those obtained by intact protein measurement. Correlation plots indicated that chymotrypsin-PRM leads to poor sensitivity and largely underestimated protein adduction levels. Trypsin-PRM leads to sensitive and highly correlated (R2 > 0.91) protein adduction levels with a detection limit below the endogenous level and relative standard deviation <25%. As a proof of concept, clinical serum samples were examined by trypsin-PRM, and a slightly higher adduction level was observed for the obesity group when compared with the healthy group. This is the first report on determining adduction levels of blood proteins for long-term exposure to CEs. The standard addition approach can be generally applied to protein adductomics with resolvable mass increments by intact protein measurement to accelerate the development of bottom-up methods close to the inherent limit.
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U2 - 10.1021/acs.analchem.9b04425
DO - 10.1021/acs.analchem.9b04425
M3 - Article
C2 - 31794208
AN - SCOPUS:85076236349
VL - 91
SP - 15922
EP - 15931
JO - Analytical Chemistry
JF - Analytical Chemistry
SN - 0003-2700
IS - 24
ER -