The α-carbon atoms of amino acids in the polypeptide backbone of proteins can react with reactive oxygen species (ROS), generating alkoxyl radicals. Subsequently, these radicals can undergo cleavage in two different ways: one via diamidation reaction, and the other through α-amidation reaction cleavage^[24], as shown in Fig. 3a, b. In the first case (Fig. 3a), the C—C bond at the radical initiation site breaks, forming a diamide structure on the side of the polypeptide fragment containing the radical, while the other side of the polypeptide fragment forms an isocyanate group. In the second case (Fig. 3b), the C—N bond at the radical initiation site breaks, converting the polypeptide fragment from the N-terminal of the protein into an amide, while the polypeptide segment from the C-terminal of the protein forms an N-α-ketoamide.

In addition, when protein side chains (especially those of proline, arginine, lysine, and threonine) are attacked by reactive oxygen species, one or more carbonyl groups can also be formed. These amino acid side chain oxidation products mainly include 2-pyrrolidone obtained from oxidation of proline residues, glutamic acid semialdehyde obtained from oxidation of arginine and proline residues, aminoadipic acid semialdehyde obtained from oxidation of lysine residues, and 2-amino-3-ketobutyric acid obtained from oxidation of threonine residues. Among these, glutamic-1-semialdehyde, the oxidation product of arginine and proline residues, and aminoadipic acid semialdehyde, the oxidation product of lysine residues, may be the two most abundant specific carbonylation products in cells.

Lipid peroxidation generates a series of reactive carbonyl compounds, including α and β-unsaturated aldehydes, di-aldehydes, and ketoaldehydes^[27], which are also known as lipid-derived electrophiles (LDEs), primarily including acrolein (ACR), malondialdehyde (MDA), and 4-hydroxy-2-nonenal (HNE), among others (Figure 6).

Taking the generation of HNE as an example, ROS attack unsaturated fatty acids to form fatty acid radicals. These radicals then react with oxygen to form peroxyl radicals (LOO·). LOO· can further react with adjacent polyunsaturated fatty acids to generate hydroperoxides and alkyl radicals, initiating a chain reaction that damages more fatty acids. LOO· may also react with neighboring double bonds, leading to cleavage of the fatty acid backbone and the formation of 4-hydroxynonenal, which is finally reduced to 4-hydroxy-2-nonenal^[28-29] (Fig. 7).

After the formation of these reactive carbonyl species, they can be conjugated onto nucleophilic amino acids such as cysteine, histidine, and lysine through Michael addition reactions or via the formation of Schiff bases^[29], thereby introducing reactive carbonyl groups into proteins and producing protein carbonylation modifications (Figure 8). Research has shown that compared to healthy individuals, the levels of lipid peroxidation compounds such as 4-hydroxy-2-nonenal and acrolein are elevated in the brains of patients with Alzheimer's disease, and increased levels of lipid peroxidation products are also observed in cerebrospinal fluid and plasma^[30-34]. Therefore, studying covalent modifications by lipid-derived electrophiles (LDEs) is not only crucial for understanding the regulatory mechanisms of electrophilic chemical signaling, but also holds significant reference value for the development of drugs targeting related diseases, such as antioxidants and targeted covalent inhibitors^[35-36].

Reactive carbonyl groups can be generated by the addition of small molecular aldehydes such as glyoxal (MG) [³⁷], methylglyoxal (MGO) [³⁸], and 3-deoxyglucosone [³⁹] (3-DG), which are produced from the degradation of glycolytic intermediates, to proteins. Additionally, the non-enzymatic Maillard reaction between amino acids and reducing sugars [⁴⁰], following a series of chemical rearrangements, can also introduce reactive carbonyl groups into protein side chains.

This article mainly focuses on the analysis and detection of protein carbonylation caused by lipid peroxidation-derived electrophiles (LDEs) resulting from reactive oxygen species (ROS) attacking lipid compounds.

As previously mentioned, the relative abundance of protein carbonylation modifications is low and their chemical stability is poor, making them difficult to analyze directly by mass spectrometry. Therefore, chemists often use specific chemical probes to derivatize and enrich carbonylated proteins before conducting mass spectrometry analysis, particularly quantitative analysis. Based on differences in their target sites, strategies for capturing protein reactive carbonyl groups can be divided into two approaches: indirect identification and direct identification. The indirect identification strategy refers to the method of indirectly identifying cysteine carbonylation modifications through a competitive reaction strategy. Specifically, proteins are first treated with LDEs capable of specifically modifying the active sites of protein cysteines; the remaining unmodified cysteine active sites are then labeled using chemical probes specific for cysteine thiol groups. At the same time, a control group without LDEs is established. Combining this with the isotopic tandem orthogonal proteolysis-activity-based protein profiling (isoTOP-ABPP) strategy and quantitative proteomics methods enables the identification of specific carbonylated proteins. Among them, the iodoacetamide alkyne probe (IA-alkyne) is an electrophilic small-molecule probe widely used in ABPP^[55]. Wang et al.^[56] developed a competitive isoTOP-ABPP strategy to quantify the reactivity of a specific LDE toward cysteines across the human proteome at a global level. The main procedure is as follows: (1) Treat one proteome sample with LDE and set up a control group treated with DMSO (dimethyl sulfoxide), followed by labeling of unmodified reactive cysteine sites using IA probes; (2) Based on click chemistry strategy (copper-catalyzed azide-alkyne cycloaddition, CuAAC), the IA-labeled cysteines in the samples are conjugated with isotopically labeled cleavable biotin-azide tags; (3) The IA-labeled proteins are enriched using streptavidin beads, followed by trypsin digestion and cleavage of the linker to release IA-alkyne-labeled peptides^[57]; (4) The samples are analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) (Figure 10). The quantitative ratio (R value, derived from LDE-treated versus untreated samples) for each cysteine-containing peptide accurately reflects the extent of LDE modification at each cysteine under a given LDE concentration. A higher R value indicates higher sensitivity to LDE modification.

The advantage of the competitive isoTOP-ABPP strategy lies in its ability to directly introduce compound molecules as competitive inhibitors into the proteome, eliminating the cumbersome chemical synthesis steps required to convert them into molecular probes^[58]. However, this method is currently limited to the analysis of LDE-cysteine interactions, making it difficult to study modifications of LDEs on histidine and lysine residues. Additionally, the application of this method is restricted due to the high cost of isotopically labeled amino acid reagents and the lengthy, low-yield synthesis processes associated with cleavable light and heavy tag reagents^[59].

Compared with indirect identification methods that rely on competitive reaction strategies, direct identification methods for protein carbonylation, which involve the direct reaction of probes with carbonyl groups at carbonylation sites or carbonylation modifications, offer higher sensitivity and can effectively reduce false positive interference. Based on the action targets of the probes, direct identification methods can also be divided into two categories: metabolic labeling and targeted direct labeling of carbonyl groups.

Metabolic labeling detects specific carbonylation types by using LDE analog probes containing "click handles" (Fig. 11). Marnett et al. introduced azide or alkyne groups into the alkyl terminus of HNE structures and used these HNE analog probes to label proteins in human colon adenocarcinoma cells. Subsequently, biotin-conjugated complementary alkynes or azides were added to perform click chemistry reactions, thereby biotinylating the target proteins. After enrichment and purification using streptavidin beads, LC-MS/MS analysis identified 538 HNE-modified proteins. Additionally, Porter et al. developed a photocleavable azide-biotin reporter tag that specifically releases HNE-alkyne probe-modified proteins or peptides from streptavidin beads after enrichment. Codreanu et al. conducted a series of quantitative modification experiments using alkynyl analogs of the lipid electrophilic reagents 4-hydroxy-2-nonenal and 4-oxo-2-nonenal to investigate the functional impacts of LDE alkylation damage. Building upon this, Liebler et al. employed isotopic labeling combined with photocleavable azide-biotin reagents to selectively capture and quantify cellular targets labeled by HNE-alkyne probes, enabling large-scale, in situ, site-specific identification and quantitative analysis of approximately 400 HNE modification sites.

ONE shares a similar structure with HNE, but their reactivities differ^[64]. Generally, HNE preferentially reacts with cysteine residues in the proteome, whereas ONE exhibits higher chemical affinity toward lysine residues. In neuronal cells, ONE demonstrates greater reactivity and cytotoxicity than HNE. Yang et al.^[65] utilized an isotopically labeled, photocleavable azido-biotin reagent to selectively capture and quantify protein targets labeled by an ONE alkynyl analog (aONE), revealing the diversity and dynamics of ONE modifications within cells through quantitative analysis.

However, metabolic labeling also has notable limitations. For example, the synthesis of specific LDEs analog probes is cumbersome and costly; it is not suitable for smaller LDE structures, such as ACR, since any chemical modification can affect its reactivity^[66].

For another direct identification method, direct labeling targeting reactive carbonyl groups, various carbonyl-reactive reagents have been developed and applied to label LDEs-modified proteins in diverse biological samples. Among these, probes based on hydrazide or hydroxylamine biotinylation are the most widely used.

The hydrazide group of biotin reacts with the active carbonyl groups formed after protein carbonylation modification to form acyl hydrazone bonds for linkage. Utilizing the high affinity of streptavidin beads, biotinylated proteins are specifically captured and enriched. Following trypsin digestion, the proteins are identified and quantified using LC-MS/MS. Soreghan et al.^[67] applied a hydrazide biotin probe combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis to identify more than 100 carbonylated proteins in the brain proteome of aged mice, including some low-abundance proteins previously missed by immunoaffinity-based methods. Codreanu et al.^[68] used this method for the first comprehensive analysis of HNE-modified proteins in the human proteome; they treated RKO cells (human colon adenocarcinoma cells) with varying concentrations of HNE, labeled the HNE-modified proteins with a hydrazide-biotin probe, enriched them using streptavidin beads, and performed LC-MS/MS analysis to identify HNE protein targets, revealing numerous important intracellular systems affected by HNE modification, including those involved in protein translation, folding, and degradation. Liu Changfeng et al. used biotin hydrazide labeling, avidin bead enrichment, and tandem mass spectrometry to isolate and identify carbonylated proteins in the striatum of aged rats and integrated bioinformatics to search for mass spectrometry identification information.^[69]

Hydroxylamine can react with active carbonyl groups to form oxime bonds. N-Aminooxymethyl carbonyl hydrazide D-biotin, also known as aldehyde-reactive probe (ARP), is a biotinylated hydroxylamine derivative. Chavez et al.^[70] used ARP to enrich target proteins and employed tandem mass spectrometry to analyze and identify HNE-labeled protein targets. This approach has been applied to proteomic studies of the human monocytic cell line THP-1^[71] and cardiac mitochondria from mouse tissues^[72], successfully identifying multiple protein targets and residue sites modified by LDE. Notably, the oxime bonds formed by hydroxylamine and active carbonyl groups exhibit significantly higher stability compared to the hydrazone bonds generated through hydrazide reactions^[73]. Furthermore, this transformation does not require an additional reduction step, simplifying the procedure. Consequently, hydroxylamine-based reaction probes (ARP) have become a preferred alternative to hydrazide biotin reagents and are widely utilized in various studies^[74-75].

However, the method of identifying carbonylated proteins using biotin probes faces issues of false positive identifications, such as certain proteins or peptides that can bind non-specifically to avidin, and some endogenous proteins that naturally carry biotin^[76-77]. To overcome the potential problem of nonspecific background proteins, Meany et al.^[78] employed stable isotope labeling of affinity-purified proteins; this strategy combines biotin hydrazide labeling, avidin affinity chromatography, and multiplex iTRAQ reagent-based stable isotope labeling techniques to enrich and identify more than 200 carbonylated proteins in rat muscle mitochondrial extracts.

Hydrazide or hydroxylamine biotinylation probes can efficiently enrich various endogenous carbonylated proteins. However, the introduction of a bulky biotin group during this process may affect the activity of small molecules, thereby influencing the probe's labeling efficiency toward target proteins^[79]. At the same time, it also increases the complexity of mass spectrometry (MS) analysis^[80].

Griffin et al.^[81] proposed a tag-free strategy, in which solid-phase hydrazide (SPH) reagent was innovatively utilized. Following enzymatic digestion of proteins, HNE-modified peptides specifically enrich through the formation of covalent yet reversible hydrazone bonds via reaction between the hydrazide immobilized on glass beads and the HNE-modified peptides. After washing away unreacted peptides, the hydrazone bonds are cleaved under acidic conditions to release the HNE-modified peptides for LC-MS/MS detection. This method avoids the addition of extra tags (e.g., biotin and fluorophores) onto aldehyde groups, facilitating the analysis of HNE modification sites. Using this approach, they identified 15 HNE-modified proteins in HNE-treated yeast. Similarly, Maier et al.^[82] combined d0/d4-succinic anhydride labeling with hydrazide-functionalized microbead (Affi-Hz gel)-based enrichment to detect, identify, and relatively quantify site-specific carbonylated proteins/peptides in biological matrices. Prokai et al.^[83] first performed differential dimethyl labeling of proteins, followed by enrichment of carbonylated peptides using solid-phase hydrazide chemistry prior to mass spectrometry analysis, enabling quantification and site identification of protein carbonylation modifications. Additionally, Lu et al.^[84] developed a method for specifically enriching HNE-modified peptides based on a fluorine derivatization probe and fluorine-based solid-phase microextraction (FSPE). The hydrophobicity of the fluorine tag significantly enhances the signal of HNE-modified peptides, while FSPE enables selective enrichment of fluorine-derivatized HNE-modified peptides directly from salt solutions and complex mixtures without requiring an additional desalting step prior to LC-MS/MS analysis, thereby simplifying the workflow.

Considering the limitations of biotinylated labeling reagents, scientists have developed bioconjugation reactions based on reactive carbonyl groups, enabling rapid and highly specific covalent labeling and forming stable covalent adducts. Based on this reaction, probes containing click chemistry handles such as alkynes were synthesized. Subsequently, biotin containing azide groups and cleavable linkers was introduced via click chemistry reactions. After enrichment, photocleavage or enzymatic cleavage to remove biotin, followed by purification, large-scale quantitative studies of derivatized carbonylated proteins or peptides were achieved.

Wang et al.^[66] developed a novel chemical proteomics approach to identify both the types and sites of proteins that can be modified by HNE (Figure 12). In this strategy, hydrazide-derived probe (HZyne) and aminooxy-derived probe (AOyne) containing bioorthogonal alkyne groups were first synthesized and compared, revealing that AOyne exhibited better labeling efficiency in HNE-treated proteomes than HZyne. Subsequently, using the AOyne probe combined with reductive dimethyl labeling technology^[86-87] and TOP-ABPP (tandem orthogonal proteolysis-ABPP) technology^[88], more than 4,000 HNE-modified proteins and approximately 400 modification sites were identified in the HEK293T cell proteome.

Wang et al.^[89] also applied aniline-derived probes to detect protein carbonylation modifications in ferroptosis, identifying more than 400 endogenous LDE-modified proteins and over 20 modification sites. They further ranked the sensitivity of these sites using the isoTOP-ABPP strategy and analyzed their roles in biological metabolic pathways through bioinformatics approaches.

Among these labeling reagents, hydrazide probes exhibit poor capture efficiency. The imine products formed by their reaction with active carbonyl groups are structurally unstable and require reduction with sodium cyanoborohydride. However, sodium cyanoborohydride shows poor selectivity during reduction, making the overall experimental procedure more complex. The capture efficiency of hydroxylamine-derived probes and aniline-derived probes is significantly affected by pH, showing notably lower efficiency under neutral or weakly basic conditions compared to acidic environments. Moreover, the products are unstable under basic conditions and tend to decompose during trypsin digestion, leading to the detachment of click handles. Therefore, the development of new labeling reagents with high efficiency, specificity, and excellent product stability remains an urgent need.