TY - JOUR
T1 - Simultaneous 3-Nitrophenylhydrazine Derivatization Strategy of Carbonyl, Carboxyl and Phosphoryl Submetabolome for LC-MS/MS-Based Targeted Metabolomics with Improved Sensitivity and Coverage
AU - Meng, Xiangjun
AU - Pang, Huanhuan
AU - Sun, Fei
AU - Jin, Xiaohan
AU - Wang, Bohong
AU - Yao, Ke
AU - Yao, Liang
AU - Wang, Lijuan
AU - Hu, Zeping
N1 - Funding Information:
We appreciate Dr. Binghui Li for providing the HeLa cells, Drs. Jianwei Wang and Min Liao for providing HSCs and K562 cells, and Drs. Wei Xie and Shuyan Ji for providing oocytes. We thank the members of the Hu laboratory for critiquing the manuscript. Z.H. is supported by grants from the National Natural Science Foundation of China (92057209), National Key R&D Program of China (2019YFA0802100-02, 2020YFA0803300), National Science and Technology Major Project for “Significant New Drugs Development” (2017ZX09304015), Tsinghua University (53332200517), Tsinghua-Peking Joint Center for Life Sciences, and Beijing Frontier Research Center for Biological Structure.
Publisher Copyright:
© 2021 American Chemical Society.
PY - 2021/7/27
Y1 - 2021/7/27
N2 - Metabolomics is a powerful and essential technology for profiling metabolic phenotypes and exploring metabolic reprogramming, which enables the identification of biomarkers and provides mechanistic insights into physiology and disease. However, its applications are still limited by the technical challenges particularly in its detection sensitivity for the analysis of biological samples with limited amount, necessitating the development of highly sensitive approaches. Here, we developed a highly sensitive liquid chromatography tandem mass spectrometry method based on a 3-nitrophenylhydrazine (3-NPH) derivatization strategy that simultaneously targets carbonyl, carboxyl, and phosphoryl groups for targeted metabolomic analysis (HSDccp-TM) in biological samples. By testing 130 endogenous metabolites including organic acids, amino acids, carbohydrates, nucleotides, carnitines, and vitamins, we showed that the derivatization strategy resulted in significantly improved detection sensitivity and chromatographic separation capability. Metabolic profiling of merely 60 oocytes and 5000 hematopoietic stem cells primarily isolated from mice demonstrated that this method enabled routine metabolomic analysis in trace amounts of biospecimens. Moreover, the derivatization strategy bypassed the tediousness of inferring the MS fragmentation patterns and simplified the complexity of monitoring ion pairs of metabolites, which greatly facilitated the metabolic flux analysis (MFA) for glycolysis, the tricarboxylic acid (TCA) cycle, and pentose phosphate pathway (PPP) in cell cultures. In summary, the novel 3-NPH derivatization-based method with high sensitivity, good chromatographic separation, and broad coverage showed great potential in promoting metabolomics and MFA, especially in trace amounts of biospecimens.
AB - Metabolomics is a powerful and essential technology for profiling metabolic phenotypes and exploring metabolic reprogramming, which enables the identification of biomarkers and provides mechanistic insights into physiology and disease. However, its applications are still limited by the technical challenges particularly in its detection sensitivity for the analysis of biological samples with limited amount, necessitating the development of highly sensitive approaches. Here, we developed a highly sensitive liquid chromatography tandem mass spectrometry method based on a 3-nitrophenylhydrazine (3-NPH) derivatization strategy that simultaneously targets carbonyl, carboxyl, and phosphoryl groups for targeted metabolomic analysis (HSDccp-TM) in biological samples. By testing 130 endogenous metabolites including organic acids, amino acids, carbohydrates, nucleotides, carnitines, and vitamins, we showed that the derivatization strategy resulted in significantly improved detection sensitivity and chromatographic separation capability. Metabolic profiling of merely 60 oocytes and 5000 hematopoietic stem cells primarily isolated from mice demonstrated that this method enabled routine metabolomic analysis in trace amounts of biospecimens. Moreover, the derivatization strategy bypassed the tediousness of inferring the MS fragmentation patterns and simplified the complexity of monitoring ion pairs of metabolites, which greatly facilitated the metabolic flux analysis (MFA) for glycolysis, the tricarboxylic acid (TCA) cycle, and pentose phosphate pathway (PPP) in cell cultures. In summary, the novel 3-NPH derivatization-based method with high sensitivity, good chromatographic separation, and broad coverage showed great potential in promoting metabolomics and MFA, especially in trace amounts of biospecimens.
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U2 - 10.1021/acs.analchem.1c00767
DO - 10.1021/acs.analchem.1c00767
M3 - Article
C2 - 34270209
AN - SCOPUS:85111341186
SN - 0003-2700
VL - 93
SP - 10075
EP - 10083
JO - Analytical Chemistry
JF - Analytical Chemistry
IS - 29
ER -