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Enrichment Strategies

MS is the key technique to identify nitropeptides and nitroproteins and to accurately locate each nitration site within the amino acid sequence of a nitro- protein [1, 2, 75]. However, because of unique MS behaviors of nitropeptides,

Table 5.2 Nitroprotein and unnitrated protein identified from pituitary adenoma [2] and control tissue [1,75].

Pituitary adenoma

Pituitary control

Protein name

nY site

Protein name

nY site

Nitrated protein

Nitrated protein

Rho-GTPase-activating 5 [Q13017] (ARHGAP5)

Y"550

Synaptosomal-associated protein (SNAP91)

Y237

Leukocyte immunoglobulin-like receptor A4 [P59901]

y404

Iga Fc receptor [P24071] (FCAR)

Y223

Zinc finger protein 432 [O94892]

y41

Actin [P03996] (ACTA2, ACTG2, ACTC1)

Y296

PKA p regulatory subunit [P31321] (PRKAR1B)

Y20

PKG 2 [Q13237] (PRKG2)

Y354

Sphingosine-1-phosphate lyase 1 [O95470]

y356

Y366

Mitochondrial cochaperone protein HscB [Q8IWL3]

Y128

Centaurin p1 [Q15027]

Y485

Stanniocalcin 1[P52823] (STC1)

Y159

Proteasome subunit a type 2 [P25787] (PSMA2)

Y228

Proteasome subunit a type 2 (PSMA2)

Y228

Interleukin 1 family member 6 [Q9UHA7] (IL1F6)

Y96

Progestin and adipoQ receptor family member III [Q6TCH7] (PAQR3)

Y33

Rhophilin 2 [Q8IUC4] (RHPN2) Nitroprotein-interacted protein

Y258

Interleukin-1 receptor-associated kinase-like 2 (IRAK-2) [O43187] (IRAK2)

Glutamate receptor interacting protein 2 [Q9C0E4] (GRIP2)

Ubiquitin [P62988] (UBB or UBC)

Source: Zhan & Desiderio, 2006 [2]. Reproduced with permission of Elsevier; Zhan & Desiderio [1]. Reproduced with permission of Elsevier; Zhan & Desiderio [75]. Reproduced with permission of Elsevier.

Note: nY = nitrotyrosine. Modified from Zhan and Desiderio [1,2,75], with permission from Elsevier Science, copyright 2004, 2006, and 2007; Reproduced from Zhan, Wang, Desiderio [38], with permission from Wiley-VCH, copyright 2015.

extremely low abundance (1 in ~106 tyrosines) of nitrotyrosine sites in an in vivo proteome [24, 25], and the limited MS sensitivity (high femtomole to low picomole level) to detect a nitropeptide [3, 9], it is necessary to isolate and preferentially enrich endogenous nitroproteins or nitropeptides before MS analysis [31, 33-35, 75].

Enrichment strategies have been developed for in vitro and in vivo nitrated samples. However, it is very important to realize that, even though most enrichment methods work well for in vitro nitrated samples, only a few enrichment methods work well for in vivo endogenous nitroproteins.

Because of easy availability of in vitro synthetic or nitrated nitropeptides or nitroproteins, some isolation and enrichment methods have been developed to analyze in vitro nitroproteins or nitropeptides in order to transit them to analyze in vivo nitroproteins and nitropeptides. Angiotensin II, ovalbumin (OVA), and BSA are the commonly used standard peptides and proteins nitrated in vitro with liquid TNM [10, 28, 29, 50, 108], peroxynitrite [109], or gaseous nitrogen dioxide and ozone (NO2 + O3) [50]. Some proteomes, for example, human plasma, were nitrated in vitro with liquid TNM and followed by nitroproteomics analysis to simulate in vivo proteome conditions [41]. In addition, because of more stable amino (-NH2) group relative to the nitro (-NO2) group during MS analysis, these nitroproteins or nitropeptides were reduced to aminoprotein or aminopep- tide with reducing agent (Na2S2O4) before MS analysis [10, 29, 110]. Most methods for nitroprotein enrichment have been developed based on immunoaffinity isolation and chemical derivation plus targeted enrichment with these prepared standard nitropeptides, nitroproteins, and nitroproteome samples in vitro, including nitroproteomic methods based on anti-3-nitrotyrosine antibody immu- nopurification and gel-based separations [28, 37] and methods that involve multidimensional chromatography, diagonal chromatography [48, 49], precursor ion scanning [28], and/or chemical derivation, which might characterize and quantify protein tyrosine nitration sites [37]. Among them, chemical derivation methods are most extensively studied in the analysis of in vitro nitropeptides and nitroproteins as modes of sample preparation before MS [36]. However, all these chemical derivation methods basically include the following procedure: reduction of the nitro to an amino group, derivation of the amino group with specific reagents, and followed by preferential enrichment. Several various strategies for chemical derivatization plus targeted enrichments have been described, including the conversion of a nitro to a more stable amino group in nitrotyrosine residue with reduction to readily distinguish aminopeptides from nonnitrated peptides with an easy-to-interpret peptide mass spectrum [10]. Due to photodecomposition of the nitro group with a MALDI UV-laser, a strategy has also been developed to acetylate N-terminal amines and e-amines of lysine residues with acetic anhydride, reduce nitro to amino groups with sodium hydrosulfite, derivatize amino groups with 1-(6-methyl[D0/D3]nicotinoyloxy) succinimide, and followed by MALDI-TOF MS analysis [43]. Improved chemical-labeling methods have been designed to enrich nitropeptides independent of sequence context. In this procedure (Figure 5.9), all amines are blocked with acetylation, followed by conversion of a nitro to an amino group and biotinylation of the resultant aminotyrosine [42].

Reaction scheme of the chemical-labeling method as exemplified with an N-terminal nitrotyrosine residue

Figure 5.9 Reaction scheme of the chemical-labeling method as exemplified with an N-terminal nitrotyrosine residue. All amines were blocked with acetylation with acetic acid N-hydroxysuccinimide ester (NHS-acetate). Nitrotyrosine was reduced to aminotyrosine with heme and DL-dithiothreitol in a boiling water bath. The reaction sequence was completed with biotinylation of aminotyrosine with NHS-biotin. Source: From Desiderio, 2015. [38] Reproduced with permission of Wiley, Abello. (2010) [42], Reproduced with permission of Elsevier.

This entire reaction can be carried out in a single buffer without any sample cleanup or pH changes to reduce sample loss. Free biotin was removed with a strong cation exchanger, labeled peptides were subsequently enriched using an immobilized avidin column, and enriched peptides were analyzed with LC-MS/ MS [42]. This method has been approved for in vitro nitrated samples [19, 42]. A method that specifically enriches nitropeptides to identify unambiguously nitropeptides and nitration sites with LC-MS/MS includes the conversion of nitrotyrosine to M-thioacetyl-aminotyrosine, followed by high-efficiency enrichment of sulfhydryl-containing peptides with thiopropyl sepharose beads [30]. Acetylation with acetic anhydride to block all primary amines, reduction of the nitro group to an amino group, derivatization of this amino group with

W-succinimidyl S-acetylthioacetate, and deprotection of the 5-acetyl on S-acetylthioacetate will subsequently form free sulfhydryl groups [30]. This method was used to study in vitro nitrated BSA, human histone H1.2, and mouse brain tissue samples [30]. An alternative and quantitative strategy, which combines precursor isotopic labeling and isobaric tagging (cPILOT), increased the multiplexing capability to quantify a nitroprotein among 12 or 16 samples with TMT or iTRAQ. For this method, light- and heavy-labeled acetyl groups were used to block N-termini and lysine residues of tryptic peptides. Reduction of a nitro to an amino group with sodium dithionite is followed by derivatiza- tion of light- and heavy-labeled aminopeptides with either multiplex TMT or iTRAQ reagents [47]. This method demonstrated proof of principle to analyze in vitro nitrated BSA and mouse splenic proteins [47]. A new strategy, also based on the reactive and quantitative properties of iTRAQ reagents coupled with MS analysis, involves selective labeling of nitrotyrosine residues [46] to simultaneously localize and quantify nitration sites in model proteins and biological systems [46]. This method overcomes the drawback of iTRAQ quantitative proteomics that is limited to primary amines. COFRADIC [48, 49], a form of diagonal chromatography, has also been employed following the reduction of the nitro to an amino group with sodium dithionite. This method sorts peptides with reversed-phase chromatography based on a hydrophilic shift from nitro- peptide (more hydrophilic) to aminopeptide (more hydrophobic) followed by ESI-MS [48] and MALDI-MS [49] identification. COFRADIC identified tyrosine nitration in a TNM-nitrated BSA and peroxynitrite-nitrated proteome of human Jurkat cells [48, 49]. Furthermore, one study has also used dansyl chloride to label nitration sites, followed by MS/MS plus a precursor ion scan [44, 45] to identify tyrosine nitration sites.

The interpretation of MS and MS/MS data of endogenous nitropeptides is very challenging. To avoid any risk of linking MS/MS spectra to an incorrect amino acid sequence, one group has combined the reduction of the nitro to an amino group and the use of the Peptizer algorithm to inspect MS/MS quality- related assumptions [111]. However, the optimal approach to determine the amino acid sequence of a nitropeptide remains a manual approach [2].

For the isolation and preferential enrichment of the much more difficult endogenous nitroproteins and nitropeptides from a biological proteome, several protocols have been developed, which are as follows: (i) two-dimensional electrophoresis (2DE)-based western blotting with antinitrotyrosine antibodies (Figure 5.10) [1, 26, 75, 112-114]. (ii) The use of nitrotyrosine affinity column (NTAC) (Figure 5.11) to enrich nitroproteins [2, 26, 86] and nitropeptides [115]. (iii) The use of COFRADIC to sort peptides according to the hydrophilic shift after the reduction of a nitro group to an amino group, followed by ESI or MALDI-MS [48]. (iv) The use of dansyl chloride to label nitration sites followed by a precursor ion scan and MS3 analysis [44, 45]. (v) After acetylation of all primary amines and reduction of nitro to amino group, derivation of amino

Two-dimensional western blotting analysis of anti-3-nitrotyrosine-positive proteins in a human pituitary

Figure 5.10 Two-dimensional western blotting analysis of anti-3-nitrotyrosine-positive proteins in a human pituitary (70 pg protein per 2D gel). (a) Silver-stained image on a 2D gel before transfer of proteins onto a PVDF membrane. (b) Silver-stained image on a 2D gel after transfer of proteins onto a PVDF membrane. (c) western blot image of anti-3- nitrotyrosine-positive proteins (anti-3-nitrotyrosine antibodies + secondary antibody).

(d) Negative control of a western blot to show the cross-reaction of the secondary antibody (only the secondary antibody; no anti-3-nitrotyrosine antibody). Source: Desiderio (2007) [75], Reproduced with permission of Elsevier Science, Zhan, Wang, Desiderio [38], Reproduced with permission of Wiley-VCH, Zhan, Wang, & Desiderio (2013) [26], reproduced with permission of Hindawi Publishing Corporation.

group into a free sulfhydryl group followed by enrichment of sulfhydryl peptides with thiopropyl sepharose beads [30]. (vi) After acetylation of all primary amines in a nitropeptide, conversion of nitro to amino group, and followed by enrichment with biotinylation of an aminotyrosine (Figure 5.9) [10, 36, 42]. (vii) The use of a new tagging reagent, (3^,4S)-1-(4-(aminomethyl)phenylsulfo- nyl) pyrrolidine-3,4-diol (APPD) for selectively fluorogenic derivation of a nitro group in nitropeptides (after reduction to aminotyrosine) followed by boronate affinity enrichment [116]. (viii) Quantitative identification of nitroproteins and

Experimental flowchart to identify nitroprotein and nitroprotein-protein complexes with NTAC-based MALDI-LTQ MS/MS

Figure 5.11 Experimental flowchart to identify nitroprotein and nitroprotein-protein complexes with NTAC-based MALDI-LTQ MS/MS. The control experiment (without any anti-3-nitrotyrosine antibody) was performed in parallel with the NTAC-based experiments. Source: Reproduced from Zhan and Desiderio [2], with permission from Elsevier Science, copyright 2006; Reproduced from Zhan et al. [38], with permission from Wiley-VCH, copyright 2015; and reproduced from Zhan et al. [26], with permission from Hindawi Publishing Corporation. Copyright 2013 remains with authors due to the open-access article under the Creative Commons Attribution License.

nitropeptides with TMT or iTRAQ [46, 47]; for this protocol, after the use of the “light”- and “heavy”-labeled acetyl groups to block N-termini and lysine residues of tryptic peptides, the nitro group was reduced to amino group and followed by derivation of light- and heavy-labeled aminopeptides with either multiplex TMT or iTRAQ reagents [46, 47]. This method can relatively enrich and quantitatively identify nitroproteins and nitropeptides.

Protocols (i)-(iii) have been used to identify endogenous nitration sites [1, 2, 48, 75, 86]. Protocols (iii)-(vii) have succeeded mainly in an in vitro nitrated peptide, protein, and proteome [10, 30, 44, 48, 49] and provide promise for studies of endogenous nitroproteins. All protocols focus on identification of nitropeptides, nitroproteins, and nitration sites. However, for discovery of disease-related nitroproteins, except for characterization of nitration sites and nitroproteins, quantitative identification of nitroproteins is needed. Protocol (viii) holds promise for that goal because it can relatively enrich nitropeptides, quantify nitroproteins, and identify nitration sites, and it has enhanced sample multiplexing capabilities [46, 47].

 
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