Determination of Total Concentration of Chemically Labeled Metabolites as a Means of Metabolome Sample Normalization and Sample Loading Optimization in Mass Spectrometry-Based Metabolomics

Abstract

For mass spectrometry (MS)-based metabolomics, it is important to use the same amount of starting materials from each sample to compare the metabolome changes in two or more comparative samples. Unfortunately, for biological samples, the total amount or concentration of metabolites is difficult to determine. In this work, we report a general approach of determining the total concentration of metabolites based on the use of chemical labeling to attach a UV absorbent to the metabolites to be analyzed, followed by rapid step-gradient liquid chromatography (LC) UV detection of the labeled metabolites. It is shown that quantification of the total labeled analytes in a biological sample facilitates the preparation of an appropriate amount of starting materials for MS analysis as well as the optimization of the sample loading amount to a mass spectrometer for achieving optimal detectability. As an example, dansylation chemistry was used to label the amine- and phenol-containing metabolites in human urine samples. LC-UV quantification of the labeled metabolites could be optimally performed at the detection wavelength of 338 nm. A calibration curve established from the analysis of a mixture of 17 labeled amino acid standards was found to have the same slope as that from the analysis of the labeled urinary metabolites, suggesting that the labeled amino acid standard calibration curve could be used to determine the total concentration of the labeled urinary metabolites. A workflow incorporating this LC-UV metabolite quantification strategy was then developed in which all individual urine samples were first labeled with (12)C-dansylation and the concentration of each sample was determined by LC-UV. The volumes of urine samples taken for producing the pooled urine standard were adjusted to ensure an equal amount of labeled urine metabolites from each sample was used for the pooling. The pooled urine standard was then labeled with (13)C-dansylation. Equal amounts of the (12)C-labeled individual sample and the (13)C-labeled pooled urine standard were mixed for LC-MS analysis. This way of concentration normalization among different samples with varying concentrations of total metabolites was found to be critical for generating reliable metabolome profiles for comparison.

Metabolite Analysis and Histology on the Exact Same Tissue: Comprehensive Metabolomic Profiling and Metabolic Classification of Prostate Cancer

“…Thus, sample amount normalization to the same total concentration of metabolites is very important in order to determine the individual metabolite concentration differences among the samples8. We used an LC-UV method19 to measure the total amount of dansyl labeled metabolites in an extract to normalize individual samples. In this approach, we extracted the metabolites from a tissue and labeled the extract using 12 C-dansylation, followed by LC-UV quantification of the labeled metabolites with the use of a calibration curve of peak area of eluted metabolites vs. varying known concentrations of a labeled amino acid standard mixture19.…”

“…We used an LC-UV method19 to measure the total amount of dansyl labeled metabolites in an extract to normalize individual samples. In this approach, we extracted the metabolites from a tissue and labeled the extract using 12 C-dansylation, followed by LC-UV quantification of the labeled metabolites with the use of a calibration curve of peak area of eluted metabolites vs. varying known concentrations of a labeled amino acid standard mixture19. According to the concentration of a labeled extract, we took a proper volume of an aliquot from each 12 C-labeled extract so that the same molar amount from all individual labeled samples was taken and mixed it with the same amount of 13 C-labeled UMS.…”

“…The total amount of the dansyl labeled metabolites in a given sample was quantified using LC-UV, as described previously19 (see Supplemental Note N1 for more detail). The total amount was used to normalize the individual samples; an equal amount of 12 C-labeled individual sample was used and mixed with the same amount of 13 C-UMS.…”

Chemical Isotope Labeling LC-MS for Monitoring Disease Progression and Treatment in Animal Models: Plasma Metabolomics Study of Osteoarthritis Rat Model

“…For sample normalization, the 12 C-labeled individual samples were separately injected onto LC-UV for quantifying the total labeled metabolites in each sample based on absorption at 338 nm61. An Agilent 1290 UPLC system with a photodiode array detector (Agilent, Palo Alto, CA) and a Waters ACQUITY UPLC BEH C18 column (2.1 mm × 10 cm, 1.7 μm particle size, 130 Å pore size) were used for LC-UV.…”

Baseline urine metabolic phenotype in patients with severe alcoholic hepatitis and its association with outcome

“…Although the preparation of urine samples for analysis is simple and the concentration of many metabolites is amplified by bladder storage, the biological interpretation of data is complicated by a variation in diuresis from subject to subject. Various normalization methods have been used and published to address this issue, including the traditional use of urinary creatinine concentration, osmolality,30, 31 total useful MS signal,30 and specific gravity19, 32 as well as a combination of creatinine concentration and normalization of the MS signal20 and the determination of the total concentration of chemically labeled metabolites by using liquid chromatography‐ultraviolet 33. However, many studies do not use normalization procedures, and there is still no consensus on this point 34.…”