MS Behavior of Nitrated Peptides
MS is a key technique to identify tyrosine nitration in a protein and each modification site. However, the MS behaviors of a nitropeptide are obviously different between MALDI UV-laser MS and infrared-MALDI-Fourier transform ion cyclotron resonance mass spectrometry (IR-MALDI-FT-ICR-MS) [27-29, 56]; between MALDI UV-laser MS and ESI-MS [27-29]; among different fragmentation models, including collision-induced dissociation (CID), electron transfer dissociation (ETD), electron capture dissociation (ECD), and metastable atom-activated dissociation (MAD)-MS [57-59]; and among different types of CID-MS/MS instruments. The various MS behaviors complicate the interpretation of MS and MS/MS spectra of a nitropeptide. Recognition of these various MS behaviors of a nitropeptide can assist in accurate identification of tyrosine nitration in a peptide.
For MALDI UV-laser MS, a photochemical decomposition pattern ([M+H]+, [M+H-16]+, [M+H - 30]+, and [M+H - 32]+) of the nitro group (-NO2) is induced by the high-energy laser at 337 nm to decrease the signal intensity of the precursor ion of a nitropeptide and complicate a MALDI-MS spectrum [27-29, 32]. Figure 5.1 summarizes the production of dityrosine and nitrotyrosine and likely products of photochemical decomposition of a nitrotyrosine .
Evidence from several experiments strongly supports this photochemical decomposition pattern of a nitro group with MALDI UV-laser and the complicated MS spectrum. Studies of a synthetic nitropeptide AAFGY(-NO2)AR ([M+H]+ = 800.4) with MALDI UV-Laser MS  found a photochemical decomposition pattern ([M+H]+, [M+H-16]+, [M+H-14]+, [M+H-32]+, and [M+H-30]+) in the MS spectrum that corresponded to m/z 800.4, 784.4, 786.4, 768.4, and 770.4, respectively (Figure 5.2).
The [M+H]+ ion (m/z 800.4) represents the nitrotyrosine (Tyr-NO2)-containing peptide; [M+H-16]+ (m/z 784.4) the nitrosotyrosine (Tyr-NO)-containing peptide after loss of an oxygen atom from a nitro group; [M+H-14]+ (m/z 786.4) the hydroxylaminotyrosine (Tyr-NHOH)-containing peptide after reduction of the nitroso (Tyr-NO) group; [M+H-32]+ (m/z 768.4) the triplet nitrenetyrosine (Tyr-N)-containing peptide after loss of two oxygen atoms; and [M+H-30]+ (m/z 770.4) the aminotyrosine (Tyr-NH2)-containing peptide after reduction of triplet nitrene (Tyr-N) group. The -30/32 Da photodecomposition products (Tyr-NH2 and Tyr-N) were obviously lower in abundance than the corresponding [M+H]+ ion (Tyr-NO2) and the -14/16 Da photodecomposition products
Figure 5.1 Generation of dityrosine and nitrotyrosine and likely products from nitrotyrosine photochemical decomposition. Source: Turko & Murad 2005 , Reproduced with permission of Elsevier, Desiderio . Reproduced with permission of Wiley.
Figure 5.2 Photodecomposition pattern of the synthetic nitropeptide AAFGY(-NO2)AR in the MALDI-TOF spectrum in the (a) linear mode and (b) reflectron mode. The structure of 3-nitrotyrosine and the proposed photodecomposition products are shown next to various ions. Several small ions (asterisk) might represent metastable peaks (see text for details).
A slight increase in the abundance of the ion at m/z 771.4 over what would be expected for the 13C isotope peak for the aminotyrosine products at m/z 770.4 in the linear and reflectron spectra suggests that a small amount of a catechol product might have formed as well. Source: Sarver 2001, . Reproduced with permission of Elsevier, Desiderio, 2015.  Reproduced with permission of Wiley.
(Tyr-NHOH and Tyr-NO). These MALDI UV-induced photodecompositions were also confirmed with nitropeptides from tetranitromethane (TNM)- nitrated bovine serum albumin (BSA) , TNM-treated angiotensin II ([M+H]+, m/z 1092.5) , and synthetic peptides, including leucine enkephalin (LE1: Y-G-G-F-L, molecular weight (MW) = 555.1818 Da), nitro-Tyr-leucine enkephalin [LE2: (3-NO2)Y-G-G-F-L, MW = 600.0909 Da], and d5-Phe-nitro- Tyr-leucine enkephalin [LE3: (3-NO2)Y-G-G-(d5)F-L, MW = 605.1818 Da] . The base peak intensity of the [M+H]+ ion of leucine enkephalin (LE1, NL = 1.01E5) was much higher than that of nitro-Tyr leucine enkephalin (LE2, NL = 3.25E4) and d(5)-Phe-nitro-Tyr leucine enkephalin (LE3, NL = 9.09E4) to demonstrate that photochemical decomposition decreased ion intensity and complicated the MS spectrum (Figure 5.3) .
For vMALDI-MS/MS analysis of LE1, LE2, and LE3, b- and a-ions were the most intense fragment ions compared with y-ions (Figure 5.4) ; these data
Figure 5.3 MALDI-MS spectra of LEI (a), LE2 (b), and LE3 (c). nY = nitro-Tyr. F(d5) = Phe residue with five 2H (d) atoms. Source: Reproduced from Zhan and Desiderio , with permission from Elsevier Science, copyright 2009; Reproduced from Zhan et al. , with permission from Wiley-VCH, copyright 2015; and reproduced from Zhan et al. , with permission from Hindawi Publishing Corporation. Copyright 2013 remains with authors due to the open-access article under the Creative Commons Attribution License.
Figure 5.4 MS2 spectra of LEI (a), LE2 (b), and LE3 (c). nY = nitro-Tyr. F(d5) = Phe residue with five 2H (d) atoms. Source: Reproduced from Zhan and Desiderio , with permission from Elsevier Science, copyright 2009; Reproduced from Zhan et al. , with permission from Wiley-VCH, copyright 2015; and reproduced from Zhan et al. , with permission from Hindawi Publishing Corporation. Copyright 2013 remains with authors due to the open- access article under the Creative Commons Attribution License.
have been corroborated with MALDI-MS/MS analysis of nitrated angiotensin II .
Compared with the unmodified peptide (LE1), more collision energy was required for optimized fragmentation of the nitropeptide (Figure 5.5a) but increased the intensity of the a4-ion and decreased the intensity of the b4-ion (a-ion = loss of CO from a b-ion) (Figure 5.5b).
Furthermore, optimized laser fluence maximized fragmentation of the nitropeptide. Although MS3 analysis confirmed the MS2-derived amino acid sequence, MS3 analysis requires a higher amount of peptide relative to MS2 . Thus, MS3 analysis might not be suitable for routine analysis of endogenous low-abundance nitroproteins. Only when a target is determined can MS3 be used for confirmation.
Figure 5.5 Effect of collision energy on fragmentation of nitropeptides. (a) Relationship between collision energy and product ion intensity (n = 3). (b) Relationship between collision energy and product ion b4 and a4 intensities (n = 3). Source: Reproduced from Zhan and Desiderio , with permission from Elsevier Science, copyright 2009; Reproduced from Zhan et al. , with permission from Wiley-VCH, copyright 2015; and reproduced from Zhan et al. , with permission from Hindawi Publishing Corporation. Copyright 2013 remains with authors due to the open-access article under the Creative Commons Attribution License.
To detect a nitropeptide, the amount of peptide must reach the sensitivity of a mass spectrometer; for synthetic nitropeptides, the sensitivity of vMALDI-LTQ was 1 fmol for MS detection and 10 fmol for MS2 detection . The precise reason why MALDI with laser light at 337 nm induces photodecompositions of a nitro group in a nitropeptide - but not with infrared light - and the structures of these decomposition products remain unknown ; however, it is probable that the higher energy content at 337 nm induces loss of one or two oxygen atoms of the nitro group in a nitropeptide . These photochemical decomposition patterns with a MALDI laser can confirm the presence of nitrotyrosine residue in the analyzed sample. IR-MALDI-FT-ICR- MS is a highly efficient method to determine protein nitration but does not fragment [M+H]+ ions of nitrotyrosine peptides . Moreover, for MALDI- MS analysis of a nitropeptide, the optimum matrix is sinapinic acid but not 2,5-dihydroxybenzoic acid .
For ESI-MS, no chemical decomposition of a nitro group is found in a spectrum [27-29, 31-35] as confirmed with ESI-MS analysis of TNM-nitrated angiotensin II [DRVY(-NO2)IHPF; MW = 1090.76 Da] . A mononitrated angiotensin II ion ([M+2H]2+ m/z 546.38, [NO2-Tyr]-angiotensin II) and dinitrated angiotensin II ion ([M+2H]2+ m/z 568.85, [(NO2)2-Tyr]-angiotensin II) are also found in the ESI-MS spectrum without decomposition (Figure 5.6).
The fragmentation of [M+2H]2+ precursor ions for mononitrated angiotensin II at m/z 546.38 and for dinitrated angiotensin II at m/z 568.85 (Figure 5.7) demonstrates characteristic immonium ions at m/z 181.06 for mononitrated tyrosine and at m/z 226.0 for dinitrated tyrosine in an ESI-MS/MS spectrum (Figure 5.7), which indicate the presence of a nitrotyrosine residue.
Figure 5.6 ESI-MS spectrum of nitrated angiotensin II to show mono- and dinitrated angiotensin II. Source: Desiderio, 2015.  Reproduced with permission of Wiley.
Figure 5.7 The MS2 spectra of nitrated angiotensin II peptides. The doubly charged ions were selected as precursor ions for mononitrated angiotensin II at m/z 546.30 (a) and for the dinitrated angiotensin II at m/z 568.80 (b). Source: Desiderio, 2015.  Reproduced with permission of Wiley, Petersson 2001. . Reproduced with permission of Wiley.
The characteristic immonium ion-based precursor ion scan spectrum accurately identifies nitropeptides in complex sample (Figure 5.8). The ESI-MS behavior of a nitropeptide and the precursor ion scans for an immonium ion at m/z 181.06 were further confirmed with ESI-MS analysis of TNM-nitrated BSA .
For MS/MS analysis of a nitropeptide, different fragmentation modes result in different fragmentation behaviors of precursor ions of a nitropeptide. Fragmentation methods include CID, ECD, ETD, and MAD [57-59]. Nitration does not appear to affect the CID behavior of peptides. However, for doubly charged peptides, production of ECD sequence fragments is severely inhibited with nitration, although ECD of triply charged nitrotyrosine-containing peptides produces some singly charged sequence fragments. ECD of nitropeptides was characterized by multiple losses of small neutral species, including hydroxyl radicals, water, and ammonia. The origin of neutral losses was investigated with activated ion (AI) ECD. Loss of ammonia appears to be the result
Figure 5.8 Precursor ion scan spectra of nitrated angiotensin II for the formation of immonium ion at m/z 181.06 for mononitrated tyrosine (a) and at m/z 226.0 for dinitrated tyrosine (b). Source: Desiderio, 2015.  Reproduced with permission of Wiley, Petersson 2001. . Reproduced with permission of Wiley.
of noncovalent interactions between a nitro group and protonated lysine side chains [57, 58]. Further studies have found that high kinetic energy helium MAD produced extensive backbone fragmentation with significant retention of post-translation modifications (PTMs). Although the high electron affinity of a nitrotyrosine moiety quenched radical chemistry and fragmentation in ECD and ETD, MAD does produce numerous backbone cleavages in the vicinity of the nitration. Compared with CID, MAD produced more fragment ions and differentiated I/L residues in nitrated peptides. MAD induced radical ion chemistry, even in the presence of strong radical traps, therefore offers unique advantages to ECD, ETD, and CID to determine a nitropeptide .