Abstract
The overproduction of reactive oxygen and nitrogen species (ROS and RNS) can have deleterious effects in the cell, including structural and possible activity-altering modifications to proteins. Peroxynitrite is one such RNS that can result in a specific protein modification, nitration of tyrosine residues to form nitrotyrosine, and to date, the identification of nitrotyrosine sites in proteins continues to be a major analytical challenge. We have developed a method by which 15N-labeled nitrotyrosine groups are generated on peptide or protein standards using stable isotope-labeled peroxynitrite (O15NOO−), and the resulting standard is mixed with representative samples in which nitrotyrosine formation is to be measured by mass spectrometry (MS). Nitropeptide MS/MS spectra are filtered using high mass accuracy Fourier transform MS (FTMS) detection of the nitrotyrosine immonium ion. Given that the nitropeptide pair is co-isolated for MS/MS fragmentation, the nitrotyrosine immonium ions (at m/z = 181 or 182) can be used for relative quantitation with negligible isotopic interference at a mass resolution of greater than 50,000 (FWHM, full width at half-maximum). Furthermore, the standard potentially allows for the increased signal of nitrotyrosine-containing peptides, thus facilitating selection for MS/MS in a data-dependent mode of acquisition. We have evaluated the methodology in terms of nitrotyrosine site identification and relative quantitation using nitrated peptide and protein standards.
Highlights
Protein modifications induced by oxidative or nitrative stress have been implicated in the pathophysiological mechanisms underlying the progression of certain diseases [1,2,3,4,5,6,7,8,9]
The heavy ONOO− can be reacted with protein standards, cell lysates or tissue protein extracts that are representative of the sample in which nitration will be measured
This protein mixture containing heavy-labeled nitration serves as an internal standard that is spiked into the sample where endogenous nitration will be identified and quantified (Figure 1b)
Summary
Protein modifications induced by oxidative or nitrative stress have been implicated in the pathophysiological mechanisms underlying the progression of certain diseases [1,2,3,4,5,6,7,8,9]. If signal-to-noise and detection specificity can be enhanced to facilitate mass spectrometry (MS)-based detection of nitrotyrosine-containing peptides, nitration site identification from complex biological matrices could be feasible using standard MS data acquisition methods, providing an alternative strategy that complements chemical derivatization and enrichment techniques that have been employed prior to MS-based analysis of low-abundance post-translational modifications (PTM), such as PTN [15,16,17]. Given the stronger signal observed for the tyrosine immonium ion, it has been shown to be a useful marker for the detection of tyrosine-targeted modifications, such as phosphorylation or nitration using precursor ion scanning [20,24,25] Some limitations of this approach are the potential isobaric interference of the nitrotyrosine immonium ion by other fragment ions at low mass resolution and the lack of sensitivity and specificity required to detect endogenous tyrosine nitration sites using precursor ion scanning on a chromatographic timescale. We describe the development and assessment of this mass spectrometry-based method for nitrotyrosine identification and relative quantitation using peptide and protein standards for evaluation
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