Efficient Identification and Quantification of Peptides Containing Nitrotyrosine by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry after Derivatization

Abstract

Protein nitration at tyrosine residues leading to 3-nitrotyrosine (NO 2 -Tyr) is one of the nonenzymatic post-translational modifications that can occur in cells under conditions of nitrosative stress.1) There are two main pathways of protein nitration. One is the generation of peroxynitrite (ONOO Ϫ ) by the reaction of nitric oxide ( · NO) with superoxide (O 2 · Ϫ ) and the subsequent reaction of ONOO Ϫ with CO 2 , which leads to the generation of nitrogen dioxide ( · NO 2 ) and the carbonate radical (CO 3 · Ϫ).2) The other is the generation of · NO 2 by the reaction of nitrite (NO 2 Ϫ ) with hydrogen peroxide (H 2 O 2 ) in the presence of heme proteins, such as myeloperoxidase.3) Tyrosine nitration can alter protein structure and function by forming NO 2 -Tyr, partly because of the shift in the pK a value of the tyrosine hydroxyl group from 10.1 to 7.2. 4) Tyrosine nitration may also prevent tyrosine phosphorylation, which is important in signal transduction. 5)Increased levels of protein nitration have been observed in various diseases, [6][7][8][9][10][11][12] such as cardiovascular disease , 9) Alzheimer's disease, 10,12) and Parkinson's disease. 11) Because protein nitration is associated with both physiological and pathological conditions, it is necessary to develop efficient methods to identify the nitration sites and quantify the nitration levels in proteins.NO 2 -Tyr is a sufficiently stable modification to be used as a biomarker of · NO/ONOO Ϫ -related damage. Several methods have been developed to detect protein nitration.12-16) Immunodetection with antibodies directed against nitrotyrosine is one of the most commonly used methods.12) Many highperformance liquid chromatography (HPLC)-based methods with ultraviolet, 13) electrochemical, 14) and fluorescent detection 15) have also been reported. However, these methods are based on the detection and/or quantification of free nitrotyrosine, either in plasma or generated after the cleavage of the peptide bond by enzymatic or acidic hydrolysis.16) These methods yield quantitative data only regarding the changes in nitration levels in whole tissues or whole proteins, and do not identify the sites of NO 2 -Tyr in proteins.Recently, mass spectrometry (MS)-based methods have been developed as an important technique for the identification of nitrated proteins and nitrated sites in proteins. Mass spectrometers with matrix-assisted laser desorption/ionization (MALDI) [17][18][19] or electrospray ionization (ESI) [20][21][22][23][24][25] have been widely used in these studies. The application of tandem mass spectrometry (MS/MS) makes it possible to identify nitrated sites in proteins. [20][21][22][23][24][25] In the MALDI analysis of nitrotyrosine-containing peptides, the characteristic mass pattern derived from nitrotyrosine has mass values lower than that of [MϩH] ϩ by 16 Da and 32 Da. These ions correspond to the loss of one and two oxygen atoms, respectively, from the nitro group (-NO 2 ), and are generated during the ionization process with laser irradiat...

Relative quantitation of protein nitration by liquid chromatography–mass spectrometry using isotope-coded dimethyl labeling and chemoprecipitation

“…We have recently reported the use of light- and heavy-isotopic N-dimethyl labeling of aliphatic amines in peptides combined with chemoprecipitation-based enrichment for relative quantification of posttranslational protein carbonylation by 4-hydroxy-2-nonenal [22]. Others have used 13 C 0 / 13 C 4 - or d 0 /d 6 -acetic anhydride for the labeling of aliphatic amines to quantify nitropeptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry [32]. However, acylation is not selective, as O-acylation also occurs [33], and it also alters peptide charge and, therefore, it may significantly reduce ionization when ESI is used for MS analysis [34].…”

Proteomic Approaches to Analyze Protein Tyrosine Nitration

“…Many studies (1,6,8,14,17,19,44) rely on the disappearance of mass spectral peaks for the starting materials to demonstrate a complete conversion for each reaction. However, this is not a truly quantitative technique, especially when MALDI is used, and furthermore this data cannot account for sample losses (e.g., due to adsorption on plastic or glassware) or undetected side products.…”

“…Electrospray ionization (ESI) avoids this problem but does have some disadvantages, such as a lower tolerance for sample contaminants and matrix components. Regardless of the ionization source, both 3NY-and 3AY-containing peptides can have low ionization efficiency and/or poor MS/MS fragmentation (1,13,17,19,28,44). 3AY may give deviating isotopic envelopes for fragment ions, possibly due to chemical rearrangements, which can interfere with protein database searching and prevent proper identification (13).…”

Proteomics quantification of protein nitration

“…Generally, precursor labeling uses the following major steps to make quantitative tagging selective for 3NT sites: 1) digest proteins to peptides, 2) block primary amines with acetyl (or other) groups, 3) reduce 3NT to 3AT, and 4) derivatize 3AT with a light or heavy isotope chemical tag. Figure 4 A highlights the incorporation of a light or heavy (e.g., 2 H 3 ) 1-(6-methylnicotinoyloxy) succinimide reagent to 3AT residues (Tsumoto et al 2010 ). The structure of this reagent contains a pyridine ring with a methyl group in the para position that has hydrogen atoms that can be exchanged for deuterium.…”