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Comparative Study
. 2011 Oct 19;74(11):2510-21.
doi: 10.1016/j.jprot.2011.04.007. Epub 2011 Apr 15.

Confident Identification of 3-nitrotyrosine Modifications in Mass Spectral Data Across Multiple Mass Spectrometry Platforms

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Free PMC article
Comparative Study

Confident Identification of 3-nitrotyrosine Modifications in Mass Spectral Data Across Multiple Mass Spectrometry Platforms

Bensheng Li et al. J Proteomics. .
Free PMC article

Abstract

3-nitrotyrosine (3NT) is an oxidative posttranslational modification associated with many diseases. Determining the specific sites of this modification remains a challenge due to the low stoichiometry of 3NT modifications in biological samples. Mass spectrometry-based proteomics is a powerful tool for identifying 3NT modifications, however several reports identifying 3NT sites were later demonstrated to be incorrect, highlighting that both the accuracy and efficiency of these workflows need improvement. To advance our understanding of the chromatographic and spectral properties of 3NT-containing peptides we have adapted a straightforward, reproducible procedure to generate a large set of 3NT peptides by chemical nitration of a defined, commercially available 48 protein mixture. Using two complementary LC-MS/MS platforms, a QTOF (QSTAR Elite) and dual pressure ion trap mass spectrometer (LTQ Velos), we detected over 200 validated 3NT-containing peptides with significant overlap in the peptides detected by both systems. We investigated the LC-MS/MS properties for each peptide manually using defined criteria and then assessed their utility to confirm that the peptide was 3NT modified. This broad set of validated 3NT-containing peptides can be utilized to optimize mass spectrometric instrumentation and data mining strategies or further develop 3NT peptide enrichment strategies for this biologically important, oxidative posttranslational modification.

Figures

Figure 1
Figure 1. Overview of experimental approach to generate, indentify, and validate 3NT peptides
An equimolar mixture of 48 proteins, UPS1, was TNM or mock treated to generate a set of nitrated and control samples. Samples were trypsin digested and analyzed with by both QSTAR Elite (QTOF) and LTQ Velos (dual ion trap) LC-MS/MS systems. Data was searched with Mascot and the each resulting 3NT peptide was analyzed by manual inspection using the criteria defined in Table 1.
Figure 2
Figure 2. Peptides from UPS1 detected by LC-MS/MS after TNM or mock treatment
4.5 pmol of UPS1 either TNM (1 h) or mock treated was trypsin digested and analyzed using both a QTOF (QSTAR Elite) and dual ion trap (LTQ Velos) mass spectrometer. Three technical replicates were acquired. A) The total number of peptides indentified including ones not containing a Y, the number of peptides with no 3NT modified Ys, and the number of peptides with 3NT modified Ys are shown. B) The proportion of 3NT modified peptides shown as a percent of all Y containing peptides detected shown for both conditions. Distinct peptides were defined as differences in the site of nitration, missed cleavages, and methionine oxidation state
Figure 3
Figure 3. LC-MS/MS analysis the nitrated and unmodified peptide YLYEIAR and its unmodified counterpart by the QSTAR Elite and LTQ Velos
A) MS/MS of the unmodified peptide YLYEIAR (MH22+, 464.3 m/z) from albumin detected by the QSTAR Elite. B) The MS/MS spectra of the 3NT modified peptide Y(NO2)LY(NO2)EIAR (MH22+, 509.2 m/z) from the QSTAR Elite and C) LTQ Velos. The Y and 3NT immonium ions are highlighted in bold as well as the y5 and b2 ions which are increased in mass due to the presence of the 3NT modification. Inset: Extracted ion chromatogram of the nitrated and unmodified peptide demonstrating the increased retention time that occurs after 3NT modification.
Figure 4
Figure 4. Overlap of validated 3NT peptides identified by the QSTAR Elite and LTQ Velos
Venn diagram of the overlap of all validated 3NT (A) and multiply 3NT modified (B) peptides identified by the QSTAR Elite and LTQ Velos. Distinct peptides were defined as differences in the site of nitration, missed cleavages, and methionine oxidation state
Figure 5
Figure 5. LC-MS/MS properties of the 3NT peptides identified by the QSTAR Elite and LTQ Velos
A) The percent of all the validated 3NT peptides which met each of the criteria assessed using Table 1 for both the QSTAR Elite and LTQ Velos mass spectrometers. B) The difference in chromatographic retention time (Δ RT) between 3NT modified peptides and their unmodified counterparts. C) Distribution of the precursor mass error (in ppm) for 3NT peptides indentified by the QSTAR Elite.
Figure 6
Figure 6. UPS1 nitration is reproducible and stable
A) Two independent nitration reactions were performed on UPS1 and the levels of nitration was evaluated by quantitative MRM. The y-axis represents ion percentage of 3NT peptide relative to the unmodified peptide with representative peptides shown. Error bars represent the standard of deviation. B) Storage stability evaluation of 3NT modified UPS1 peptides by quantitative MRM assay of a set of ions on 4000 QTRAP MS platform. The y-axis represents the peak area percentage of the 3NT peptide divided by the total peak area of the unmodified and 3NT species of the peptide. The x-axis displays storage period in month at -20°C.

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