Quantitative Analysis Of Amino Metabolites In Hemolymph Of Silkworm,Bombyx Mori

There are many amino-containing metabolites in the organisms,including biogenic amines,amino acids,amino acid derivatives,catecholamines,aromatic amines and so on.These amino metabolites play an important role in the organisms.The determination of amino metabolites can reflect different physiological and pathological state of the organisms,which is very significance for disease diagnosis,physiological and pathological studies.Targeted Metabolomics is a method of quantitative analysis of known metabolites with the advantage from which directly extracting biological information.Targeted metabolomics,basing on liquid chromatography-mass spectrometry,can be used to qualitatively and quantitatively analyze different metabolites by selecting suitable separation methods,ionization methods and mass analyzers.We developed a new UHPLC-MS/MS method for simultaneous quantification of the amino-group containing metabolites based on derivatization-assisted sensitivity enhancement by 5-aminoisoquinolyl-N-hydroxysuccinimidyl carbamate(5-AIQC).By using an N-ethylmaleimide-based click reaction followed with addition of antioxidants(TCEP and ascorbic acid),our method enabled simultaneous quantification of thiols,disulfides and oxidation-prone metabolites concurrently with other amino analytes in an one-pot manner.This method is also applicable to quantify aromatic amines which cannot be done with the previously reported 6-AQC-based method.This 5-AIQC-based method had higher sensitivity than the 6-AQC-based one for an extensive coverage of analytes including 4 amino saccharides,20 proteinogenic amino acids,57 non-proteinogenic amino acids,17 modified amino acids,26 aliphatic and 8 aromatic amines,22 sulfur-containing analytes,14 monoamine neurotransmitters and 8 small peptides.Amongst them,many sets of isomeric analytes(or having the same ion)were also separable on a common reversed-phase column and quantifiable with tandem-mass spectrometry.This method enables simultaneous quantification of 124 important functional metabolites in more than twenty metabolic pathways such as protein biosynthesis/degradation,biosynthesis of arginine,glutathione and catecholamine neurotransmitters,urea cycle,uridine catabolism,polyamine pathway.Our method had excellent precision,accuracy,linearity and recovery for most of analytes including thiols,disulfides and other oxidation-prone metabolites in standards,renal tumor extracts,rat urine and serum samples.We further applied this newly developed method to analyze the amino metabolites in the silkworm haemolymph at various developmental stages with 45 amino metabolites quantified.These included 20 proteinogenic and 11 non-proteinogenic amino acids,6 sulfur-containing metabolites,2 polyamines,2 catecholamines and 2 ethanolamines.Amongst them,many amino metabolites were few reported in the classical studies of silkworm haemolymph including GSSG,4-Hydroxy-L-proline,1-Methyl-L-histidine,3-Methyl-L-histidine,L-2-aminoadipic acid,y-Aminobutyric acid,L-2-Aminobutyric acid,DL-3-Aminoisobutyric acid,DL-methionine sulfoxide,DL-Methionine sulfone,Putrescine,1,3-Diaminopropane,Dopamine,3,4-Dihydroxy-DL-phenylalanine.The concentration of these amino metabolites varied greatly during different developmental stages of the silkworm,and the level of each amino metabolite was also different.The concentrations of common protein amino acids generally more than 1mM,and changes in accordance with the different biological requirements during different developmental stages.The concentrations of most amino acids was increased in the molting stage,and decreased after molting,generally increased at pre-pupal stage.The results showed that the concentrations of amino acid metabolites in silkworm(A)and hybrid silkworm(B and C)hemolymph had similar levels druing different developmental periods.However,there are significant differences in the concentration of some amino metabolites which are involved in the main physiological activities of silkworm(such as pigment synthesis,silk protein synthesis,proline energy metabolism,etc).Furthermore,in the three silkworms methionine sulfoxide derived from oxidation of methionine was about two orders of magnitude higher at the pre-pupa stage than the earlier developmental stages probably resulting from degradation of proteins to prepare for further development.5-AIQC-based method will be particularly useful for screening large cohort samples.With many classes of metabolites simultaneously quantified in an one-pot manner,this method may have useful potentials in clinical chemistry settings as well.