N-type inactivation and the S4-S5 region of the Shaker K+ channel

The intracellular segment of the Shaker K+ channel between transmembrane domains S4 and S5 has been proposed to form at least part of the receptor for the tethered N-type inactivation "ball." We used the approach of cysteine substitution mutagenesis and chemical modification to test the importance of this region in N-type inactivation. We studied N-type inactivation or the block by a soluble inactivation peptide ("ball peptide") before and after chemical modification by methanethiosulfonate reagents. Particularly at position 391, chemical modification altered specifically the kinetics of ball peptide binding without altering other biophysical properties of the channel. Results with reagents that attach different charged groups at 391 C suggested that there are both electrostatic and steric interactions between this site and the ball peptide. These findings identify this site to be in or near the receptor site for the inactivation ball. At many of the other positions studied, modification noticeably inhibited channel current. The accessible cysteines varied in the state-dependence of their modification, with five- to tenfold changes in reactions rate depending on the gating state of the channel.     

Microplate filtration assay for nicotinamide release from NAD using a boronic acid resin

We describe a microplate-based assay for NAD-dependent Class III histone deacetylases (also known as SIRTs) that measures the enzyme-catalyzed release of nicotinamide from radiolabeled NAD, using a boronic acid resin to selectively capture the NAD. This method avoids the need for fluorogenic or radiolabeled peptides or separation of the reaction products using solvent extraction. The protocol reported here is rapid and uses commercially available materials. The use of a simple microplate filtration device allows for the simultaneous processing of 96 samples, facilitating enzyme kinetic analyses and inhibition studies. Furthermore, monitoring nicotinamide release rather than peptide deacetylation obviates the need for chemical modification of protein and peptide substrates. This assay is applicable to SIRTs and other enzymes that cleave nicotinamide from NAD.     

A proteomics grade electron transfer dissociation-enabled hybrid linear ion trap-orbitrap mass spectrometer

Here we detail the modification of a quadrupole linear ion trap-orbitrap hybrid (QLT-orbitrap) mass spectrometer to accommodate a negative chemical ionization (NCI) source. The NCI source is used to produce fluoranthene radical anions for imparting electron transfer dissociation (ETD). The anion beam is stable, robust, and intense so that a sufficient amount of reagents can be injected into the QLT in only 4-8 ms. Following ion/ion reaction in the QLT, ETD product ions are mass-to-charge (m/z) analyzed in either the QLT (for speed and sensitivity) or the orbitrap (for mass resolution and accuracy). Here we describe the physical layout of this device, parametric optimization of anion transport, an evaluation of relevant ETD figures of merit, and the application of this instrument to protein sequence analysis. Described proteomic applications include complex peptide mixture analysis, post-translational modification (PTM) site identification, isotope-encoded quantitation, large peptide characterization, and intact protein analysis. From these experiments, we conclude the ETD-enabled orbitrap will provide the proteomic field with several new opportunities and represents an advance in protein sequence analysis technologies.     

Ames Salmonella/mammalian-microsome testing of peptides and peptide synthesis reagents

The Ames Salmonella/mammalian-microsome assay was used to evaluate the bacterial mutagenicity of 6 bioactive peptides and of 11 chemical reagents used in peptide synthesis. Samples of 2 reagents, bis(2-oxo-3-oxazolidinyl)phosphinic chloride and fluoren-9-ylmethyl chloroformate, showed mutagenic activity with strains TA100 and TA1535, and with TA1537, respectively. No mutagenic activity was found with the bioactive peptides or with the other 9 peptide synthesis reagents.     

Influence of amino acid side-chain modification on the uptake system for beta-lactam antibiotics and dipeptides from rabbit small intestine

The influence of chemical modification of functional amino acid side-chains in proteins on the H(+)-dependent uptake system for orally active alpha-amino-beta-lactam antibiotics and small peptides was investigated in brush-border membrane vesicles from rabbit small intestine. Neither a modification of cysteine residues by HgCl2, NEM, DTNB or PHMB and of vicinal thiol groups by PAO nor a modification of disulfide bonds by DTT showed any inhibition on the uptake of cephalexin, a substrate of the intestinal peptide transporter. In contrast, the Na(+)-dependent uptake systems for D-glucose and L-alanine were greatly inhibited by the thiol-modifying agents. With reagents for hydroxyl groups, carboxyl groups or arginine the transport activity for beta-lactam antibiotics also remained unchanged, whereas the uptake of D-glucose and L-alanine was inhibited by the carboxyl specific reagent DCCD. A modification of tyrosine residues with N-acetylimidazole inhibited the peptide transport system and did not affect the uptake systems for D-glucose and L-alanine. The involvement of histidine residues in the transport of orally active alpha-amino-beta-lactam antibiotics and small peptides (Kramer, W. et al. (1988) Biochim. Biophys. Acta 943, 288-296) was further substantiated by photoaffinity labeling studies using a new photoreactive derivative of the orally active cephalosporin cephalexin, 3-[phenyl-4-3H]azidocephalexin, which still carries the alpha-amino group being essential for oral activity. 3-Azidocephalexin competitively inhibited the uptake of cephalexin into brush-border membrane vesicles. The photoaffinity labeling of the 127 kDa binding protein for beta-lactam antibiotics with this photoprobe was decreased by the presence of cephalexin, benzylpenicillin or dipeptides. A modification of histidine residues in brush-border membrane vesicles with DEP led to a decreased labeling of the putative peptide transporter of Mr 127,000 compared to controls. This indicates a decrease in the affinity of the peptide transporter for alpha-amino-beta-lactam antibiotics by modification of histidine residues. The data presented demonstrate an involvement of tyrosine and histidine residues in the transport of orally active alpha-amino-beta-lactam antibiotics across the enterocyte brush-border membrane.     

In situ maleimide bridging of disulfides and a new approach to protein PEGylation

The introduction of non-natural entities into proteins by chemical modification has numerous applications in fundamental biological science and for the development and manipulation of peptide and protein therapeutics. The reduction of native disulfide bonds provides a convenient method to access two nucleophilic cysteine residues that can serve as ideal attachment points for such chemical modification. The optimum bioconjugation strategy utilizing these cysteine residues should include the reconstruction of a bridge to mimic the role of the disulfide bond, maintaining structure and stability of the protein. Furthermore, the bridging chemical modification should be as rapid as possible to prevent problems associated with protein unfolding, aggregation, or disulfide scrambling. This study reports on an in situ disulfide reduction-bridging strategy that ensures rapid sequestration of the free cysteine residues in a bridge, using dithiomaleimides. This approach is then used to PEGylate the peptide hormone somatostatin and retention of biological activity is demonstrated.     

Sequence homology analysis of a heterogenous protein population by chemical and enzymic digestion using a two-dimensional sodium dodecyl sulfate-polyacrylamide gel system

A procedure for examining possible sequence homology between two or more proteins in a heterogenous protein mixture using a two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis system is described. Three different chemical reagents (cyanogen bromide, hydroxylamine, and acetic acid) and three enzymes (α-chymotrypsin, trypsin, and Staphylococcus aureus protease) have been used as the cleavage reagents for the peptide mapping studies. Potential application of this technique in conjunction with radioactive labeling and immunological studies was also demonstrated.     

Localization of the strychnine binding site on the 48-kilodalton subunit of the glycine receptor

Amino acid residues that participate in antagonist binding to the strychnine-sensitive glycine receptor (GlyR) have been identified by selectively modifying functional groups with chemical reagents. Moreover, a region directly involved with strychnine binding has been localized in the 48-kDa subunit of this receptor by covalent labeling and proteolytic mapping. Modification of tyrosyl or arginyl residues promotes a marked decrease of specific [3H]strychnine binding either to rat spinal cord plasma membranes or to the purified GlyR incorporated into phospholipid vesicles. Occupancy of the receptor by strychnine, but not by glycine, completely protects from the inhibition caused by chemical reagents. Furthermore, these tyrosine- or arginine-specific reagents decrease the number of binding sites (Bmax) for [3H]strychnine binding without affecting the affinity for the ligand (Kd). These observations strongly suggest that such residues are present at, or very close to, the antagonist binding site. In order to localize the strychnine binding domain within the GlyR, purified and reconstituted receptor preparations were photoaffinity labeled with [3H]strychnine. The radiolabeled 48-kDa subunit was then digested with specific chemical proteolytic reagents, and the peptides containing the covalently bound radioligand were identified by fluorography after gel electrophoresis. N-Chlorosuccinimide treatment of [3H]strychnine-labeled 48K polypeptide yielded a single labeled peptide of Mr approximately 7300, and cyanogen bromide gave a labeled peptide of Mr 6200.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of new biotin/streptavidin reagents for pretargeting

The high affinity of biotin for streptavidin has made this pair of molecules very useful for in vivo applications. To optimize reagents for one potential in vivo application, antibody-based pretargeting of cancer, we have prepared a number of new biotin and streptavidin derivatives. The derivatives developed include new radiolabeled biotin reagents, new protein biotinylation reagents, and new biotin multimers for cross-linking and/or polymerization of streptavidin. We have also modified streptavidin by site-directed mutation and chemical modification to improve its in vivo characteristics, and have developed new reagents for cross-linking antibody fragments with streptavidin. A brief overview of these new reagents is provided.     

Tyrosine residues modification studied by MALDI-TOF mass spectrometry

Amino acid residue-specific reactivity in proteins is of great current interest in structural biology as it provides information about solvent accessibility and reactivity of the residue and, consequently, about protein structure and possible interactions. In the work presented tyrosine residues of three model proteins with known spatial structure are modified with two tyrosine-specific reagents: tetranitromethane and iodine. Modified proteins were specifically digested by proteases and the mass of resulting peptide fragments was determined using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. Our results show that there are only small differences in the extent of tyrosine residues modification by tetranitromethane and iodine. However, data dealing with accessibility of reactive residues obtained by chemical modifications are not completely identical with those obtained by nuclear magnetic resonance and X-ray crystallography. These interesting discrepancies can be caused by local molecular dynamics and/or by specific chemical structure of the residues surrounding.     

ANIBAL, stable isotope-based quantitative proteomics by aniline and benzoic acid labeling of amino and carboxylic groups

Identification and relative quantification of hundreds to thousands of proteins within complex biological samples have become realistic with the emergence of stable isotope labeling in combination with high throughput mass spectrometry. However, all current chemical approaches target a single amino acid functionality (most often lysine or cysteine) despite the fact that addressing two or more amino acid side chains would drastically increase quantifiable information as shown by in silico analysis in this study. Although the combination of existing approaches, e.g. ICAT with isotope-coded protein labeling, is analytically feasible, it implies high costs, and the combined application of two different chemistries (kits) may not be straightforward. Therefore, we describe here the development and validation of a new stable isotope-based quantitative proteomics approach, termed aniline benzoic acid labeling (ANIBAL), using a twin chemistry approach targeting two frequent amino acid functionalities, the carboxylic and amino groups. Two simple and inexpensive reagents, aniline and benzoic acid, in their (12)C and (13)C form with convenient mass peak spacing (6 Da) and without chromatographic discrimination or modification in fragmentation behavior, are used to modify carboxylic and amino groups at the protein level, resulting in an identical peptide bond-linked benzoyl modification for both reactions. The ANIBAL chemistry is simple and straightforward and is the first method that uses a (13)C-reagent for a general stable isotope labeling approach of carboxylic groups. In silico as well as in vitro analyses clearly revealed the increase in available quantifiable information using such a twin approach. ANIBAL was validated by means of model peptides and proteins with regard to the quality of the chemistry as well as the ionization behavior of the derivatized peptides. A milk fraction was used for dynamic range assessment of protein quantification, and a bacterial lysate was used for the evaluation of relative protein quantification in a complex sample in two different biological states.     

Chemical modification of lactose repressor protein using N-substituted maleimides.

Lactose repressor protein has been modified with N-ethylmaleimide, two N-maleimide spin labels, and an N-maleimide fluorophore. The reaction with repressor cysteine residues has been characterized. Approximately 2 of the 3 eq of cysteine/repressor monomer are reactive toward these reagents. Repressor cysteines are reactive toward these reagents in the order cysteine 140 greater than or equal to cysteine 107 greater than cysteine 281. The reaction is sulfhydryl-specific. Comparison of chemical modification data obtained in this laboratory using a variety of sulfhydryl-specific reagents has been used to assess chemical features of individual cysteine environments. Effects of the maleimide reagents on biological activity have been determined. Only the fluorophore N-(3-pyrene)maleimide has significant effect; this agent selectively perturbs repressor's ability to bind to operator DNA. This result suggests that regions of protein structure surrounding 1 or more of the cysteine residues possess determinants required for normal operator DNA binding.     

Activation by laser irradiation of a "switched on" oligonucleotide reagent for an addressed modification of nucleic acids

Properties of oligonucleotide reagents containing an alkylating group of regulated reactivity (nitrogen mustard residue activatable upon mild borohydride reduction of the aromatic formyl group) have been studied. It was shown that these reagents can also be activated by irradiation with nitrogen laser light (lambda 337 nm). Activation of the reagent in complex with a target polydeoxyribonucleotide resulted in the addressed chemical modification of the target. The positional direction of the modification depended on the way of the activation (borohydride reduction or laser irradiation).     

Sulfhydryl modification ofE. coli cpn60 leads to loss of its ability to support refolding of rhodanese but not to form a binary complex

Differential chemical modification ofE. coli chaperonin 60 (cpn60) was achieved by using one of several sulfhydryl-directed reagents. For native cpn60, the three cysteines were accessible for reaction with N-ethylmaleimide (NEM), while only two of them are accessible to the larger reagent 4,4′-dipyridyl disulfide (4-PDS). However, no sulfhydryl groups were modified when the even larger reagents 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) or 2-(4′-(iodoacetamido)anilino) naphthalene-6-sulfonic acid (IAANS), were employed, unless the chaperonin was unfolded. The cpn60 that had been covalently modified with NEM or IAANS, was not able to support the chaperonin-assisted refolding of the mitochondrial enzyme rhodanese, which also requires cpn10 and ATP hydrolysis. However, both modified forms of cpn60 were able to form binary complexes with rhodanese, as demonstrated by their ability to arrest the spontaneous refolding of the enzyme. That is, chemical modification with these sulfhydryl-directed reagents produced a species that was not prevented from interaction with partially folded rhodanese, but that was prevented from supporting a subsequent step(s) during the chaperonin-assisted refolding process.     

To tag or not to tag: a comparative evaluation of immunoaffinity-labeling and tandem mass spectrometry for the identification and localization of posttranslational protein carbonylation by 4-hydroxy-2-nonenal, an end-product of lipid peroxidation

Posttranslational carbonylation of proteins by the covalent attachment of the lipid peroxidation product 4-hydroxy-2-nonenal (HNE) is a biomarker of oxidative stress. Tandem mass spectrometry (MS/MS) has become an essential tool for characterization of this modification. Chemical tagging methods have been used to facilitate the immunoaffinity-based enrichment or even quantification of HNE-modified peptides and proteins. With MS/MS spectra of the untagged modified peptides considered as references, a comparative evaluation is presented focusing on the impact of affinity-tagging with four carbonyl-specific reagents (2,4-dinitrophenyl hydrazine, biotin hydrazide, biotinamidohexanoic acid hydrazide and N'-aminooxymethylcarbonyl-hydrazino D-biotin) on collision-induced dissociation of the tagged HNE-carbonylated peptides. Our study has shown that chemical labeling may not be carried out successfully for all the peptides and with all the reagents. The attachment of a tag usually cannot circumvent the occurrence of strong neutral losses observed with untagged species and, in addition, fragmentation of the introduced tag may also happen. Chemical tagging of certain peptides may, nevertheless, afford more sequence ions upon MS/MS than the untagged carbonylated peptide, especially when Michael addition of the lipid peroxidation product occurs on cysteine residues. Therefore, tagging may increase the confidence of identifications of HNE-modified peptides by database searches.     

Design and synthesis of visible isotope-coded affinity tags for the absolute quantification of specific proteins in complex mixtures

Identification of proteins in complex mixtures by mass spectrometry is most useful when quantitative data is also obtained. We recently introduced isotope-coded affinity tags (ICAT reagents) for the relative quantification of proteins present in two or more biological samples. In this report, we describe a new generation of ICAT reagents that contain the following additional features: (1) a visible tag that allows the electrophoretic position of tagged peptides during separation to be easily monitored; (2) a photocleavable linker that allows most of the tag to be removed prior to mass spectrometric analysis; (3) an isotope tag that contains carbon-13 and nitrogen-15 atoms instead of deuterium to ensure precise comigration of light and heavy tagged peptides by reverse-phase HPLC. These reagents contain an iodoacetyl group that selectively reacts with peptide cysteine residues. Peptide modification chemistry is also reported that allows tagging of peptides that are devoid of cysteine. The synthesis of these visible isotope-coded affinity tags (VICAT reagents), and their reaction with peptides are described in this report. VICAT reagents containing a carbon-14 visible probe or an NBD fluorophore are described. These reagents are most useful for the determination of the absolute quantity of specific target proteins in complex protein mixtures such as serum or cell lysates.     

The selective blocking of the polymerization reaction of striated muscle actin leading to a derivative suitable for crystallization. Modification of Tyr-53 by 5-diazonium-(1H)tetrazole.

The polymerization reaction of rabbit muscle actin was completely inhibited by reaction of one amino acid side chain per protein monomer with 5-diazonium-(1H)[14C]tetrazole. A tryptic peptide fingerprint showed a single peptide labeled by the reagent. The peptide was isolated and the labeled amino acid identified by amino acid analysis as Tyr-53. This side chain is not accessible to the reagent in F-actin. The modification is compared to similar inhibitions by other reagents.     

Chemical modification of Salmonella typhimurium phosphoribosylpyrophosphate synthetase with 5'-(p-fluorosulfonylbenzoyl)adenosine. Identification of an active site histidine

Liquid chromatographic procedures have been developed for rapidly locating the site of reaction of chemical modification reagents with Salmonella typhimurium 5-phosphoribosyl-alpha-1-pyrophosphate (PRPP) synthetase. The enzyme was reacted with the active site-directed reagent 5'-(p-fluorosulfonylbenzoyl)adenosine (FSBA). FSBA bound to the enzyme with an apparent KD of 1.7 +/- 0.4 mM. The enzyme was inactivated during the reaction, and a limiting stoichiometry of 1.2 mol of FSBA/mol of enzyme subunit corresponded to complete inactivation. Inclusion of ATP in the reaction protected the enzyme from inactivation and incorporation of the reagent. Inclusion of ribose 5-phosphate increased the rate of reaction of PRPP synthetase with FSBA. Amino acid analyses of acid hydrolysates of modified enzyme failed to detect any known FSBA-amino acid adducts. Tryptic digestion of 5'-(p-fluorosulfonylbenzoyl)-[3H]adenosine-modified enzyme at pH 7.0 yielded a single radioactive peptide. The peptide, TR-1, was subjected to combined V8 and Asp-N protease digestion, and a single radioactive peptide was isolated. This radioactive peptide yielded the sequence Asp-Leu-His-Ala-Glu, which corresponded to amino acid residues 128-132 in S. typhimurium PRPP synthetase. No radioactivity was associated with any of the phenylthiohydantoin-amino acid fractions, all of which were recovered in good yield. A majority of the radioactivity was found in the waste effluent (64%) and on the glass fiber filter loaded into the sequenator (23%). The lability of the modification and the sequence of this peptide indicate His130 as the site of reaction with FSBA.     

Evaluation of geranylazide and farnesylazide diphosphate for incorporation of prenylazides into a CAAX box-containing peptide using protein farnesyltransferase.

Protein farnesyltransferase (PFTase) catalyzes the attachment of a geranylazide (C10) or farnesylazide (C15) moiety from the corresponding prenyldiphosphates to a model peptide substrate, N-dansyl-Gly-Cys-Val-Ile-Ala-OH. The rates of incorporation for these two substrate analogs are comparable and approximately twofold lower than that using the natural substrate farnesyl diphosphate (FPP). Reaction of N-dansyl-Gly-Cys(S-farnesylazide)-Val-Ile-Ala-OH with 2-diphenylphosphanylbenzoic acid methyl ester then gives a stable alkoxy-imidate linked product. This result suggests future generations whereby azide groups introduced using this enzymatic approach are functionalized using a broad range of azide-reactive reagents. Thus, chemistry has been developed that could be used to achieve highly specific peptide and protein modification. The farnesylazide analog may be useful in certain biological studies, whereas the geranylazide group may be more useful for general protein modification and immobilization.