GC–MS characterization of the derivatives of Lys and its PTM metabolites of methylation and hydroxylation
Derivatization of amino acids (AA) and their metabolites first with 2 M HCl/CH3OH and then with PFPA/EA yields the methyl ester (Me) N-pentafluoropropionyl (PFP) derivatives (d0Me)m-AA-(PFP)n, where m is the number of esterified carboxyl groups and n is the number of the PFP-acylated amine groups. The general formula for the amino acids derivatives prepared with 2 M HCl/CD3OD and subsequently with PFPA/EA is (d3Me)m-AA-(PFP)n. GC–MS spectra were generated from separately derivatized and analysed amino acids and their metabolites. The same GC–MS conditions were used including the oven temperature program. The structures of the derivatives were elucidated on the basis of their mass spectra, the expected 3-Da difference between d0Me and d3Me per each carboxylic group in corresponding ions, and the expected shorter retention time (tR) of the deuterium-containing derivatives. As Lys was of particular interest in our study, the results from Lys and its metabolites are separately summarized in Table 1.
Due to its single carboxylic group and the two amine groups, the derivatization of unlabelled and labelled Lys (K) yielded (d0Me)1-K-(PFP)2 and (d3Me)1-K-(PFP)2. The derivatives eluted at 9.48 min and 9.46 min, and their calculated molecular masses (M) are 452 and 455, respectively. The most intense ions in their GC–MS spectra were m/z 432 [M-HF]− and m/z 435 [M-HF]− due to neutral loss each of a HF (20 Da) group from the molecular anions [M]− at m/z 452 at m/z 455, respectively (see supplementary Fig. S1). The GC–MS spectra of the derivatives contained further mass fragments which differed by 3 Da and other mass fragments which did not differ in their m/z values (Table 1).
The derivatization of synthetic Nε-methyl-l-lysine Lys, i.e., NεMK, yielded (d0Me)1-NεMK-(PFP)2 (M, 466) and (d3Me)1-NεMK-(PFP)2 (M, 469) which eluted at 9.77 min and 9.75 min, respectively, i.e., behind the derivatives of Lys. The most intense anions were m/z 446 and m/z 449 ([M-HF]−), respectively (Table 1), whereas no molecular anions [M]− were present in both mass spectra (Fig. 2). The derivatization of synthetic Nα-methyl-l-lysine Lys, i.e., NαMK, yielded (d0Me)1-NαMK-(PFP)2 (M, 466) and (d3Me)1-NαMK-(PFP)2 (M, 469) which eluted at 10.35 min and 10.34 min, respectively, i.e., considerably later than the derivative of the isomeric Nε-methyl-l-lysine. The GC–MS spectra of (d0Me)1-NαMK-(PFP)2 and (d3Me)1-NαML-(PFP)2 contained the most intense ions at m/z 188 and less intense mass fragments differing by 3 Da, with m/z 220 and m/z 223 being the most intense (Fig. 3, Table 1). Thus, Fig. 2 and Fig. 3 indicate that the isomeric N-methyl-lysine metabolites NεMK and NαMK can be discriminated by GC–MS when analyzed as their methyl ester pentafluoropropionyl derivatives.
The derivatization of Nε-acetyl-l-lysine (NεAcK) and Nα-acetyl-l-lysine (NαAcK) under the same conditions was associated with abundant decomposition, with the main derivatives being those of lysine i.e., (d0Me)1-K-(PFP)2) (not shown). The mass spectrum of the GC–MS peak of Nε-acetyl-l-lysine (NεAcK) was about 100 times less intense than that of the lysine derivative. The mass spectrum of the GC–MS peak of Nα-acetyl-l-lysine (NαAcK) was even about 1000 times less intense than that of the lysine derivative (Fig. 4, Table 1). These results indicate that Nε-acetyl-l-lysine is considerably more stable than the Nα-acetyl-l-lysine. Because the derivatives are separated chromatographically, Nε-acetyl-l-lysine could be better quantifiable in biological samples than Nα-acetyl-l-lysine.
The derivatization of Nε,Nε-dimethyl-l-lysine (NεNεMK) resulted in very small peaks with retention times of about 8.08 min of which the GC–MS spectra are shown in Fig. 5 and Table 1. The most intense mass fragments m/z 188 and m/z 168 were common to the non-deuterated and deuterated methyl esters and are not specific for the NεNεMK derivatives as they were also observed in the GC–MS spectra of the Nα-methyl-l-lysine derivatives. The GC–MS spectra of d0Me-NεNεMK-PFP and d3Me-NεNεMK-PFP contain less intense mass fragments which differ by 3 Da, i.e., m/z 314 and m/z 317, and m/z 214 and m/z 217, and are therefore suitable for quantitative analysis of Nε,Nε-dimethyl-l-lysine (Fig. 5). Nε-Methyl-l-lysine and Nε,Nε-dimethyl-l-lysine were found not to decompose to l-lysine. The derivatization of synthetic Nε,Nε,Nε-trimethyl-l-lysine did not result in any derivative extractable into toluene. This observation suggests that Nε,Nε,Nε-trimethyl group is stable under the derivatization conditions and, because of the permanent positive charge of Nε,Nε,Nε-trimethyl-l-lysine, even formation of d0Me-NεNεNεMK-PFP and d3Me-NεNεNεMK-PFP will not allow extraction of these derivatives into toluene or any other water-immiscible organic solvents compatible with GC–MS.
5-Hydroxy-l-lysine is an enzymatic PTM metabolite of Lys residues in proteins. The most common 5-hydroxylysine stereoisomer found in collagen is 5(R)-hydroxy-l-lysine (Gjaltema and Bank 2017). The Me-PFP derivatives of synthetic 5(S,R)-hydroxy-l-lysine eluted as almost baseline-separated peaks of comparable size at 8.55 min and 8.66 min for unlabelled and 8.53 min and 8.64 min for the deuterium-labelled derivatives and had virtually identical GC–MS spectra (Fig. S2, Table 1). Several ions differed by 3 Da thus allowing quantitative analyses of both isomers. The GC–MS results strongly suggest that 5-hydroxy-lysine is converted to its methyl ester tris-pentafluoropropionyl derivatives d0Me-5-hydroxy-Lys-(PFP)3 and d3Me-5-hydroxy-Lys-(PFP)3. As Me-PFP derivatives of racemic amino acids are chromatographically not separated in our system (Hanff et al. 2019), the elution of the racemic d0Me-5-hydroxy-Lys-(PFP)3 and d3Me-5-hydroxy-Lys-(PFP)3 as double almost baseline-separated peaks suggests that the separation is due to the derivatized 5-hydroxy group of 5-hydroxy-l-lysine.
GC–MS characterization of the derivatives of l-Lys-derived AGEs
As AGEs derived from Lys, Arg and Cys are carboxylic acids and contain amine groups, we tested the utility of GC–MS for their quantitative analysis as Me-PFP derivatives using in situ prepared or commercially available stable-isotope labelled analogs. The results are summarized in Table 2 and in Figs. 6, 7, 8, 9 and 10.
GC–MS analysis of the unlabelled and deuterium-labelled Nε-(1-carboxymethyl)-l-lysine (NεCMK) derivatives resulted in the formation of closely eluting GC–MS peaks around 11.4 min without evidence of decomposition to Lys. The mass spectra of these GC–MS peaks are shown in Fig. 6 and Table 2 and can be reliably assigned to the dimethyl esters–dipentafluoropropionyl derivatives: (d0Me)2-NεCMK-(PFP)2 (M, 524) and (d3Me)2-NεCMK-(PFP)2 (M, 530), (d0Me)2-NεCd3MK-(PFP)2 (M, 527) and (d3Me)2-NεCd3MK-(PFP)2 (M, 533).
GC–MS analysis of unlabelled and deuterium-labelled Nε-(1-carboxyethyl)-l-lysine (NεCEK) resulted in the formation of the derivatives of l-lysine, suggesting decomposition of NεCEK at its Nε amine group to form the Lys derivative. The most intense GC–MS peaks from NεCEK derivatization were observed at 11.37 min and 11.33 min (Fig. 7, Table 2). These GC–MS spectra suggest formation of dimethyl ester-dipentafluoropropionyl derivatives of NεCEK, i.e., (d0Me)2-NεCEK-(PFP)2 (M, 538) and (d3Me)2-NεCEK-(PFP)2 (M, 544). These results suggest that the methyl group of NεCEK on its terminal carboxylic greatly contributes to its instability during the derivatization processes.
Nε-(2-Furoylmethyl)-l-lysine (NεFMK, furosine) is the AGE of l-lysine with fructose. Derivatization of synthetic NεFMK resulted in the formation of a single GC–MS peak eluting at about 13.4 min. This is the longest retention time of Lys and its metabolites (Tables 1, 2). There was no formation of the Lys derivative during the derivatization steps suggesting a stable NεFMK derivative at the Nε-(2-furoylmethyl) residue (data not shown). The most intense mass fragments were m/z 451 for the unlabelled NεFMK and m/z 454 for the deuterium-labelled NεFMK (Fig. 8, Table 2).
GC–MS characterization of the derivatives of Arg- and Cys-derived AGEs
Under the same derivatization procedures and GC–MS conditions, the derivatives of synthetic NG-(1-carboxymethyl)-l-arginine and NG-(1-carboxyethyl)-l-arginine eluted as very small GC–MS peaks that contained each a single intense mass fragment (Fig. 9, Table 2). Interestingly, the carboxyethyl derivatives of Arg and Lys eluted in front of the corresponding carboxymethyl derivatives. No derivatives of Arg were observed from derivatized NG-(1-carboxymethyl)-l-arginine and NG-(1-carboxyethyl)-l-arginine suggesting a higher stability of the NG-(1-carboxymethyl)-l-arginine and NG-(1-carboxyethyl)-l-arginine derivatives compared to the corresponding Lys derivatives.
S-(2-Succinyl)-l-cysteine (SC) derivatization resulted in elution each of a relatively large GC–MS peak at 12.18 min (unlabelled) and 12.13 min (labelled) of which the spectra contained mass fragments differing by 3 Da, 6 Da and 9 Da (Fig. 10). These results suggest formation of (d0Me)3-SC-(PFP)1 and (d3Me)3-SC-(PFP)1, respectively. Derivatization of the commercially available (S-carboxymethyl)-l-cysteine (CMC) yielded a small GC–MS peak eluting at about 10 min (Fig. S3). A very small GC–MS peak was obtained from derivatized (S-carboxyethyl)-l-cysteine (CEC) eluting at 11.8 min (Fig. S4, Table 2). These results indicate decomposition during the derivatization steps.
Method validation in human urine
The GC–MS method was validated using 10-µL aliquots of a pooled human urine sample in relevant concentration ranges for all analytes on three consecutive days in triplicate for each concentration. After sampling for the first validation day, the pooled urine sample was frozen at − 20 °C. This procedure was repeated on the next two days. The respective internal standards were in situ prepared using a mixture of the unlabelled analytes and derivatization in 2 M HCl/CD3OD to finally achieve concentrations being within the respective ranges of the analytes. Of the final toluene extracts, 1-µL aliquots were injected split-less and selected-ion monitoring (SIM) was performed. The same dwell-time was used for each analyte and its internal standard. The peak areas of analytes and internal standard were used for quantification. The concentration of each analyte was calculated by multiplying the PAR of analyte-to-internal standard and by the known concentration of the respective internal standard. The precision of the method was expressed as the relative standard deviation (RSD, %) from the triplicate analyses. The accuracy of the method was determined for the added analyte concentrations by subtracting the respective endogenous (basal) concentration in the un-spiked urine sample, dividing the difference with the respective added concentration and multiplying the outcome by 100. The accuracy of the method was expressed as recovery (%). Linear regression analysis was performed between measured analyte concentration (y) and added analyte concentration (x). The y-axis intercept of the regression equation provides the mean analyte concentration in the urine sample used in method validation. The slope value of the regression equation multiplied by 100 yields the mean recovery value for the analyte in the investigated concentration range. In total, the concentrations of 33 amino acids and their metabolites were determined simultaneously. Table 3 lists the SIM pairs used in quantitative analyses including the method validation.
The results from the method validation for all analytes are listed in Table S1 in the Supplement to this article. For the sake of simplicity and clarity, the results of the validation method for Lys, Arg, Cys and their PTM metabolites and AGEs are presented in Table 4.
The precision of the method ranged between 0.27% and 17.9%. The accuracy of the method ranged between 76.3 and 112%. The lowest recovery values (76.2%, 90%, 83.5%) were obtained for CMC, notably on method validation on day #1. The lower recovery values for CMC could be in part due to the low intensity of the mass fragments used in the validation. The selected mass fragments were used to achieve higher specificity in quantitative measurements. The correlation coefficients (r2) ranged between 0.9837 and 0.999. The validation results indicate that the GC–MS method is useful for the precise and accurate measurement of low concentrations of the PTM metabolites and AGEs in the presence of considerably higher concentrations of the parent amino acids in human urine. The results of Table S1 confirm the validity of the GC–MS method for other amino acids (Hanff et al. 2019). Thus, the GC–MS method presented here is suitable for the simultaneous quantitative determination of amino acids and their PTM metabolites and AGEs.
In the human urine sample used in method validation, S-(2-succinyl)-l-Cys (S2C) and 5-hydroxy-l-Lys (5-OH-Lys, 2nd GC–MS peak) were found to be the most abundant metabolites followed by CML, CEL and CEA. It appears from Table 1 that the concentrations of the analytes in the un-spiked urine samples increased after 1 and 2 days (Table 4). Yet, the concentrations of all measured amino acids also increased compared to the first day of validation, on average by 5% on day #2 and by 11% on day #3 (Fig. S5).
Typical GC–MS chromatograms from the simultaneous quantitative analysis of amino acids, their PTM metabolites and AGEs in a human urine sample are shown in Fig. S6.
Stability of the amino acid derivatives in toluene extracts
As toluene extracts may not always be analyzed immediately after derivatization and extraction by toluene (Baskal et al. 2021b), we randomly selected the toluene extracts of seven different urine samples and analyzed by GC–MS freshly obtained toluene extracts (day #1). After completion of the first analysis run, the toluene extracts were analyzed again on next day (day #2) under the same GC–MS conditions. Then, the autosampler samples were sealed again and left stand at room temperature until renewed analysis one week later (day #8). This procedure was repeated one more time and the toluene extracts were analyzed again one week later (day #15). Statistical analysis of the concentrations of the analytes revealed reproducible results for Lys, Arg, and their PTM metabolites and AGEs (Table S2). The peak areas of the endogenous analytes and their internal standards did not change remarkably suggesting that the amino acid derivatives are stable for at least two weeks, thus allowing reliable quantitative determination in human urine samples at least within two weeks after sample derivatization. The highest reproducibility values were observed for 5-hydroxy-lysine, lysine, monomethyl-lysine, arginine, ADMA, succinyl-cysteine and furosine (relative standard deviation, RSD, 1.5–4.7%), with the other analytes showing poorer yet acceptable reproducibility (RSD, 8.9–15.2%) (Table S2).
Amino acids and their PTM metabolites and AGEs in boys’ urine of the ASOS study
The validated GC–MS method was applied to measure the concentration of PTM metabolites and AGEs of Lys, Arg and Cys as well as of other amino acids in urine samples of 39 black boys and 41 white boys of the ASOS study. As the urine samples were collected by spontaneous micturition, we also measured the creatinine concentrations in the ASOS urine samples, and corrected the urinary excretion of the amino acids by the respective creatinine concentration. The results of these analyses are summarized in Table 5. The urinary creatinine concentrations did not differ between black and white boys (15.3 [10.1–21.4] mM vs. 15.9 [12.8–18.8] mM, P = 0.504).
Statistically significant differences between black and white boys were found for the creatinine-corrected excretions of Thr and hArg (lower in the blacks), Asn/Asp, Pro, and Arg (higher in the blacks). The excretion rates of OH-Pro and 5-OH-Lys-D were higher in the black compared to the white boys. The excretion rates of MML and CMC were lower in the black compared to the white boys. The greatest differences between the groups were found for MML (threefold in the whites). ROC curve analyses showed that of all analytes (range 0.50–0.66) only CMC and MML were associated with high area values: 0.840 for CMC and 0.796 for MML with P < 0.0001 (Fig. S7). The excretion rates of furosine and MMA failed significant differences (Table 5). Only in the white group, a single correlation between the urinary excretion rate of hArg and age was found (r = 0.375, P = 0.016).