Mass spectrometric characterization of lipid‐modified peptides for the analysis of acylated proteins

Hoffman, Michael D.; Kast, Juergen

doi:10.1002/jms.981

Cited by 41 publications

(40 citation statements)

References 45 publications

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“…Differences in acyl chain length between lipid substrates were documented by 28-Da shifts in a series of corresponding b and y ions. Comparison of fragmentation patterns of differently modified peptides is especially important because thioester modifications often do not produce a marker or neutral loss ions; therefore backbone fragmentation appears to be the primary way of peptide decomposition (23). In conclusion, we directly demonstrated that LRAT forms transient thioester bonds during enzymatic activity, providing proof for previous hypotheses.…”

Section: Expression and Purification Of Lrat-gst Fusionsupporting

confidence: 68%

An Acyl-covalent Enzyme Intermediate of Lecithin:Retinol Acyltransferase*

Golczak

Palczewski

2010

Journal of Biological Chemistry

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Synthesis of fatty acid retinyl esters determines systemic vitamin A levels and provides substrate for production of visual chromophore (11-cis-retinal) in vertebrates. Lecithin:retinol acyltransferase (LRAT), the main enzyme responsible for retinyl ester formation, catalyzes the transfer of an acyl group from the sn-1 position of phosphatidylcholine to retinol. To delineate the catalytic mechanism of this reaction, we expressed and purified a fully active, soluble form of this enzyme and used it to examine the possible formation of a transient acyl-enzyme intermediate. A representative member of the NlpC/P60 superfamily (1), lecithin:retinol acyltransferase (LRAT), 2 the main enzyme responsible for retinyl ester formation, catalyzes the transfer of an acyl group from the sn-1 position of phosphatidylcholine (PC) to a retinoid acceptor (2-4). LRAT plays a fundamental role in the retention and storage of vitamin A and controls the generation of all-trans-retinoic acid, a potent expression regulator of many genes via retinoic acid nuclear receptors (5, 6). Importantly, LRAT also plays a key role in the retinoid cycle by providing substrate for 11-cis-retinal production (7, 8), the chromophore required for regeneration of mammalian visual pigments (9 -11). Unlike many other acyltransferases, this single membrane-spanning protein does not require an activated fatty acyl group attached by a thioester bond to coenzyme A as a donor (12). Instead, this enzyme uses a variety of phosphatidylcholines as substrates (13,14). The proposed mechanism of LRAT catalysis involves formation of a transient covalent acylenzyme intermediate accompanied by the release of lyso-phosphatidylcholine (15, 16). A number of biochemical studies involving site-directed mutagenesis and protein labeling suggest that the LRAT reaction mechanism may involve a Cys side chain that serves as an active site nucleophile (17). However, despite the above observations, self-acylation of a protein constituting part of the catalytic acyl transfer mechanism, even though postulated for several other enzymatic reactions including lysophosphatidylcholine:lysophosphatidylcholine acyltransferase and class II tumor suppressor H-Rev107, has yet to be directly documented for this class of enzymes (18,19). In this study, we employed mass spectrometry to investigate formation of a covalent acyl-enzyme intermediate and determine the site of putative modification. Additionally, we examined the influence of changes in the acyl chain length at the sn-1 position of phosphatidylcholine and the surrounding lipid phase on LRAT enzymatic activity and formation of the enzyme-bound intermediate. EXPERIMENTAL PROCEDURESProtein Expression and Purification-Mouse LRAT cDNA encoding amino acids 30 GGG…TVK 186 was subcloned into a pGEX_2T vector (GE Healthcare) using BamHI and EcoRI restriction sites to generate the glutathione S-transferase fusion protein GST-tLRAT. This fusion protein then was expressed in the Bl21(DE3) Escherichia coli strain. Bacteria were grown in the presence of ...

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Section: Expression and Purification Of Lrat-gst Fusionsupporting

confidence: 68%

An Acyl-covalent Enzyme Intermediate of Lecithin:Retinol Acyltransferase*

Golczak

Palczewski

2010

Journal of Biological Chemistry

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“…This is because there is an inverse correlation between the prevalence of the neutral loss and the charge state of the precursor ion that was observed in the product ion analysis of the sulfinamide modified-test peptides (Fig. 2) as well as in a previous study (52). If the modification has a low proton affinity and the molecular ion is in a low charge state, the probability that the modification fragments as a neutral fragment increases.…”

Section: Global Proteome Analysis For Discovery Of Hno-induced Modifisupporting

confidence: 51%

Identification of Nitroxyl-induced Modifications in Human Platelet Proteins Using a Novel Mass Spectrometric Detection Method

Hoffman

Walsh

Rogalski

et al. 2009

Molecular & Cellular Proteomics

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Nitroxyl (HNO) exhibits many important pharmacological effects, including inhibition of platelet aggregation, and the HNO donor Angeli's salt has been proposed as a potential therapeutic agent in the treatment of many diseases including heart failure and alcoholism. Despite this, little is known about the mechanism of action of HNO, and its effects are rarely linked to specific protein targets of HNO or to the actual chemical changes that proteins undergo when in contact with HNO. Here we study the presumed major molecular target of HNO within the body: protein thiols. Cysteine-containing tryptic peptides were reacted with HNO, generating the sulfinamide modification and, to a lesser extent, disulfide linkages with no other long lived intermediates or side products. The sulfinamide modification was subjected to a comprehensive tandem mass spectrometric analysis including MS/MS by CID and electron capture dissociation as well as an MS 3 analysis. These studies revealed a characteristic neutral loss of HS(O)NH 2 (65 Da) that is liberated from the modified cysteine upon CID and can be monitored by mass spectrometry. Upon storage, partial conversion of the sulfinamide to sulfinic acid was observed, leading to coinciding neutral losses of 65 and 66 Da (HS(O)OH). Validation of the method was conducted using a targeted study of nitroxylated glyceraldehyde-3-phosphate dehydrogenase extracted from Angeli's salt-treated human platelets. In these ex vivo experiments, the sample preparation process resulted in complete conversion of sulfinamide to sulfinic acid, making this the sole subject of further ex vivo studies. A global proteomics analysis to discover platelet proteins that carry nitroxyl-induced modifications and a mass spectrometric HNO dose-response analysis of the modified proteins were conducted to gain insight into the specificity and selectivity of this modification. These methods identified 10 proteins that are modified dose dependently in response to HNO, whose functions range from metabolism and cytoskeletal rearrangement to signal transduction, providing for Nitric oxide (NO)1 has emerged as an important physiological signaling molecule, particularly in the vascular, neuronal, and immune systems. NO regulates many processes including platelet function, vascular tone, and leukocyte recruitment mainly through the cGMP second messenger system (1). More recent studies have shown that nitric oxide can react directly with a number of different biological species including metal centers of proteins, nucleophilic amino acid residues (nitrosation/S-nitrosylation), and aromatic amino acid residues (nitration) (2, 3), and the products of these reactions have been analyzed by mass spectrometry (4 -8). The biological relevance of these reactions is slowly coming to light, and NO-mediated S-nitrosylation has now been linked to a number of diseases including diabetes, multiple sclerosis, cystic fibrosis, and asthma (9).Nitroxyl (HNO/NO Ϫ ), an alternative redox form of NO, has only recently begun to draw attention in th...

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“…Recently, also direct mass spectrometric techniques have been applied for the analysis of farnesylated peptides using MALDI-and ESI-mass spectrometry (Hoffman and Kast, 2006). While precursor ion scanning (reporter ion C 15 H 25 , m = 205 Da) is favourable for ESI-MS, for MALDI-MS neutral loss scanning (loss of C 15 H 24 , Dm = 204.3 Da) should be preferred but also peptide fragments can yield these ions, e.g.…”

Section: Protein Prenylationmentioning

confidence: 99%

“…In a recent report (Hoffman and Kast, 2006) also ESI-NLS has been used for palmitoylation of cysteines using the loss of C 16 H 30 OS (Dm = 272 Da). However, no marker ions were obtained which could have been used for PIS.…”

Section: Protein Acylationmentioning

confidence: 99%