Quantification of modified nucleotides and nucleosides by isotope dilution mass spectrometry.

Epigenetic modifications are closely related to certain disorders of the organism, including the development of tumors. One of the main epigenetic modifications is the methylation of DNA cytosines, 5-methyl-2'-deoxycycytidine. Furthermore, 5-mdC can be oxidized to form three new modifications, 5-(hydroxymethyl)-2'-deoxycytidine, 5-formyl-2'-deoxycytidine, and 5-carboxy-2'-deoxycytidine. The coupling of liquid chromatography with tandem mass spectrometry has been widely used for the total determination of methylated DNA cytosines in samples of biological and clinical interest. These methods are based on the measurement of the free compounds (e.g., urine) or after complete hydrolysis of the DNA (e.g., tissues) followed by a preconcentration, derivatization, and/or clean-up step. This review highlights the main advances in the quantification of modified nucleotides and nucleosides by isotope dilution using isotopically labeled analogs combined with liquid or gas chromatography coupled to mass spectrometry reported in the last 20 years. The different possible sources of labeled compounds are indicated. Special emphasis has been placed on the different types of chromatography commonly used (reverse phase and hydrophilic interaction liquid chromatography) and the derivatization methods developed to enhance chromatographic resolution and ionization efficiency. We have also revised the application of bidimensional chromatography and indicated significant biological and clinical applications of these determinations.

Evaluation of different isotope dilution mass spectrometry strategies for the characterization of naturally abundant and isotopically labelled peptide standards.

Natural abundance and isotopically labelled tryptic peptides are routinely employed as standards in quantitative proteomics. The certification of the peptide content is usually carried out by amino acid analysis using isotope dilution mass spectrometry (IDMS) after the acid hydrolysis of the peptide. For the validation and traceability of the amino acid analysis procedure, expensive certified peptides must be employed. In this work we evaluate different IDMS alternatives which will reduce the amount of certified peptide required for validation of the amino acid analysis procedure. In this context, the characterization of both natural and isotopically labelled synthetic angiotensin I peptides was carried out. First, we applied a fast procedure for peptide hydrolysis based on microwave-assisted digestion and employed two certified peptide reference materials SRM 998 angiotensin I and CRM 6901-b C-peptide for validation of the hydrolysis procedure. The amino acids proline, leucine, isoleucine, valine, tyrosine, arginine and phenylalanine were evaluated for their suitability for peptide certification by IDMS by both liquid chromatography with tandem mass spectrometry (LC-MS/MS) and gas chromatography with mass spectrometry (GC)-MS/MS. Then, natural angiotensin I and 13C1-labelled angiotensin I were synthesized in-house and purified by preparative liquid chromatography. The concentration of the 13C1-labelled angiotensin I peptide was established by reverse IDMS in its native form using SRM 998 angiotensin I as reference. The concentration of the natural synthesized peptide was determined by IDMS both using the 13C1-labelled peptide in its native form and by amino acid analysis showing comparable results. Finally, the synthetic naturally abundant angiotensin I peptide was employed as "in-house" standard for the validation of subsequent peptide characterization procedures. Therefore, the novelty of this work relies on, first, the development of a faster hydrolysis procedure assisted by focused microwaves, providing complete hydrolysis in 150 min, and secondly, a validation strategy combining GC-MS and LC-MS/MS that allowed us to certify the purity of an in-house-synthesized peptide standard that can be employed as quality control in further experiments.

A cost-effective approach to produce 15N-labelled amino acids employing Chlamydomonas reinhardtii CC503.

BACKGROUND: The use of enriched stable isotopes is of outstanding importance in chemical metrology as it allows the application of isotope dilution mass spectrometry (IDMS). Primary methods based on IDMS ensure the quality of the analytical measurements and traceability of the results to the international system of units. However, the synthesis of isotopically labelled molecules from enriched stable isotopes is an expensive and a difficult task. Either chemical and biochemical methods to produce labelled molecules have been proposed, but so far, few cost-effective methods have been described. RESULTS: The aim of this study was to use the microalgae Chlamydomonas reinhardtii to produce, at laboratory scale, 15N-labelled amino acids with a high isotopic enrichment. To do that, a culture media containing 15NH4Cl was used. No kinetic isotope effect (KIE) was observed. The labelled proteins biosynthesized by the microorganism were extracted from the biomass and the 15N-labelled amino acids were obtained after a protein hydrolysis with HCl. The use of the wall deficient strain CC503 cw92 mt+ is fit for purpose, as it only assimilates ammonia as nitrogen source, avoiding isotope contamination with nitrogen from the atmosphere or the reagents used in the culture medium, and enhancing the protein extraction efficiency compared to cell-walled wild type Chlamydomonas. The isotopic enrichment of the labelled amino acids was calculated from their isotopic composition measured by gas chromatography mass spectrometry (GC-MS). The average isotopic enrichment for the 16 amino acids characterized was 99.56 ± 0.05% and the concentration of the amino acids in the hydrolysate ranged from 18 to 90 µg/mL. CONCLUSIONS: Previously reported biochemical methods to produce isotopically labelled proteins have been applied in the fields of proteomics and fluxomics. For these approaches, low amounts of products are required and the isotopic enrichment of the molecules has never been properly determined. So far, only 13C-labelled fatty acids have been isolated from labelled microalga biomass as valuable industrial products. In this study, we propose Chlamydomonas reinhardtii CC503 as a feasible microorganism and strain to produce labelled biomass from which a standard containing sixteen 15N-labelled amino acids could be obtained.