Protein carbonylation has been associated with various human diseases such as Alzheimer's disease, Parkinson's disease, chronic lung disease, chronic renal failure, diabetes, sepsis and sclerosis [1
]. Generally, there are several types of amino acid oxidative modifications that can give rise to protein carbonyls [2
]. Protein carbonyl derivatives can also be formed through reactions with reactive carbonyl compounds produced during oxidative conversion of various biomolecules such as lipids [5
]. Among the reactive carbonyl compounds, 4-hydroxy-2-nonenal (HNE) has drawn particular attention and has been the most well studied lipid peroxidation end-product [6
]. HNE is formed from polyunsaturated fatty acids present in biological membranes and it reacts readily with nucleophilic groups of protein amino acid side chains. Several studies have shown that covalent attachment of HNE to proteins lead to alteration in their structure and biological activity [7
]. Modification by HNE occurs on nucleophilic side-chains of amino acid residues primarily via Michael addition or Schiff-base (imine) formation [9
]. HNE modification through Michael addition involves reaction of the imidazole group of histidine (His), the ε-amino group of lysine (Lys), or the sulfhydryl group of cysteine (Cys) with the C=C double bond of HNE (). Schiff-base is also formed by the reaction of HNE with the ε-amino group of Lys. The reactivity of amino acids toward HNE has shown to be Cys>His>Lys [11
]. Michael adducts generally represent 99% of HNE protein modifications, whereas Schiff-base adduct formation is less prevalent even in the presence of excess HNE and does not result in protein carbonylation [12
Reaction of nucleophiles in amino acid side chains by Michael addition.
Protein targets of HNE-modification have been identified by 2-D polyacrylamide gel electrophoresis in which mass spectrometry is used merely for protein identification, mostly by peptide-mass fingerprinting and, thus, without seeking modification-specific information at the peptide level [14
]. The availability of modern tandem mass spectrometers have prompted efforts to utilize them for the modification- and sequence-directed identification of carbonylation through the formation of covalent adducts with HNE. Due to the low abundance of this posttranslational modification, enrichment of HNE-modified peptides usually is required before mass spectrometric analyses [17
]. Therefore, there has been much interest recently about development of methods to enrich carbonylated proteins and peptides for mass spectrometric analyses [18
]. Solid-phase hydrazide chemistry has been employed for the enrichment of HNE-carbonylated peptides [17
]. The feature of this method is that it recovers the modified species in its native, unlabeled form and may also allow for the use of sophisticated additional chemistry enabling partial 18
O-labeling of reactive carbonyl modifications, which produces a unique isotope signature in mass spectra, to detect the modified peptides [23
]. However, the solid-phase hydrazide reagent immobilized on controlled pore glass particles is not available commercially and, hence, has to be synthesized for the study, a task that some laboratories may not be prepared to perform. Recently, affinity columns have been made by immobilizing an antibody recognizing HNE-Michael adducts, and the use of these columns also yields samples of enriched untagged peptides [24
]. However, the majority of the techniques rely on labeling (“tagging”) the carbonyl group for label-specific immunoaffinity enrichment.
When gel electrophoresis is used for putative identification of carbonylated proteins [25
], chemical derivatization of protein carbonyl groups are commonly carried out with 2,4-dinitrophenylhydrazine (DNPH, ), a carbonyl-specific tag detected by anti-DNP antibodies (“oxyblot”) [27
]. However, even this well-established procedure of redox proteomics have been scrutinized recently concerning methodological details [28
]. Nevertheless, immunoaffinity purification of DNPH-derivatized malondialdehyde- and HNE-adducts of peptides has been developed to facilitate discovery-driven exploration of these posttranslational modifications by LC–MS/MS [29
]. N’-aminooxymethylcarbonylhydrazino D-biotin, known as aldehyde-reactive probe (ARP, ), has also been used to label HNE-carbonyls for biotin-avidin affinity enrichment and subsequent identification of protein targets in complex proteomes [18
]. Additional biotin-based reagents such as biotin hydrazide (BH) [22
] or biotinamidohexanoic acid hydrazide (a long-chain hydrazide-activated biotin, LCBH) [31
] have also been applied for enrichment-enabling immunoaffinity tagging which requires an additional reduction step to prevent the loss of label in subsequent steps in the proteomics workflow, in studies directed to protein carbonylation and in conjunction with shotgun proteomic analyses. However, no evidence has been sought that tagging reactions perform uniformly for all peptides modified by HNE.
Labeling of HNE-carbonylated peptides with (a) DNPH, (b) ARP, and (c) BH and LCBH.
Collision-induced dissociation (CID) has been the prevalent method to obtain MS/MS spectra that potentially afford modification-directed identification at peptide level concerning HNE adducts. Most commonly, yet not exclusively [18
], ion-trap instruments have been used for this purpose [17
]. CID of HNE-modified peptides may, however, be confounded by the occurrence of strong neutral loss (NL) obscuring sequence ions necessary for peptide identification and localization of the site of modification [17
]. Specifically, Michael adducts of HNE to peptides may produce NL of the lipid peroxidation product (–156 Da) upon CID, albeit the degree of this NL is apparently peptide-dependent [34
]. Different chemical tagging methods have similarly revealed various extent of NLs of HNE-adducted tags [18
]. ARP-labeled HNE carbonyls, like the relatively large non-cleavable isotope-coded affinity tag (ICAT) also containing a biotin moiety [35
], display tag fragmentation upon CID [30
] and, thus, may complicate MS/MS analysis of tagged peptides and subsequent database searching [36
]. Overall, affinity tags for protein carbonylation were developed to permit selective enrichment, but their impact on MS/MS-based identification of HNE-modified peptides has not been addressed and/or evaluated.
Using selected tryptic peptides of proteins with biological importance as models [30
], we compared labeling methods and CID features of untagged and chemically tagged HNE-carbonylated peptides. In particular, we focused on identification of this posttranslational modification by liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS) on an ion trap instrument—the most common platform of discovery-driven proteomics in this field. Specifically, we evaluated four different HNE labeling methods (DNPH, ARP, BH and LCBH, as shown in ) and the fragmentation of the resultant tagged species upon CID to survey the impact of tagging on the identification of HNE-modification by LC–ESI-MS/MS analyses in the context of shotgun-based redox proteomics.