Novel intra- and inter-molecular sulfinamide bonds in S100A8 produced by hypochlorite oxidation.

Hypochlorite is a major oxidant generated when neutrophils and macrophages are activated at inflammatory sites, such as in atherosclerotic lesions. Murine S100A8 (A8) is a major cytoplasmic protein in neutrophils and is secreted by macrophages in response to inflammatory stimuli. After incubation with reagent HOCl for 10 min, approximately 85% of A8 was converted to 4 oxidation products, with electrospay ionization mass spectrometry masses of m/z 10354, 10388, 10354 +/- 1, and 20707 +/- 3. All were resistant to reduction by dithiothreitol. Initial formation of a reactive Cys sulfenic acid intermediate was demonstrated by the rapid conjugation of 5,5-dimethyl-1,3-cyclohexanedione (dimedone) to HOCl-treated A8 to form stable adducts. Matrix-assisted laser desorption-reflectron time of flight peptide mass fingerprinting of isolated oxidation products confirmed the mass additions observed in the full-length proteins. Both Met(36/73) were converted to Met(36/73) sulfoxides. An additional product with an unusual mass addition of m/z 14 (+/-0.2) was identified and corresponded to the addition of oxygen to Cys(41), conjugation to various epsilon-amines of Lys(6), Lys(34/35), or Lys(87) with loss of dihydrogen and formation of stable intra- or inter-molecular sulfinamide cross-links. Specific fragmentations identified in matrix-assisted laser desorption-post source decay spectra and low energy collisional-induced dissociation tandem mass spectroscopy spectra of sulfinamide-containing digest peptides confirmed Lys(34/35) to Cys(41) sulfinamide bonds. HOCl oxidation of mutants lacking Cys(41) (Ala(41)S100A8) or specific Lys residues (e.g. Lys(34/35), Ala(34/35)S100A8) did not form sulfinamide cross-links. HOCl generated by myeloperoxidase and H(2)O(2) and by phorbol 12-myristate 13-acetate-activated neutrophils also formed these products(.) In contrast to the disulfide-linked dimer, oxidized monomer retained normal chemotactic activity for neutrophils. Sulfinamide bond formation represents a novel oxidative cross-linking process between thiols and amines and may be a general consequence of HOCl protein oxidation in inflammation not identified previously. Similar modifications in other proteins could potentially regulate normal and pathological processes during aging, atherogenesis, fibrosis, and neurogenerative diseases.

S100 calcium-binding proteins are highly conserved, small (ϳ10 kDa) acidic proteins with important regulatory functions including regulation of kinases, suppression of tumor progression, embryogenesis, and cell migration (1)(2)(3)(4)(5). Some 18 members are described, and genes of 13 are clustered on chromosome 1q21 (6,7). S100 proteins form non-covalent homodimers characterized by a symmetric homodimeric fold not found in other Ca 2ϩ -binding proteins in solution. Dimerization is mediated by hydrophobic contacts within helix IV, residues at the C terminus of helix I, and individual residues within the extended C-terminal domain. Ca 2ϩ binding induces structural changes within helices III and IV, exposing amino acids within the hinge and C-terminal domains that may be involved in binding target proteins (5,8). Covalent oxidative modifications may regulate the functions of S100B, A8, 1 and S100A2 (9 -11).
A8/S100A9 (A9), present constitutively in high concentrations (ϳ40% of cytosolic protein) in PMN, can be released from activated or dying cells at inflammatory sites (1,32,33), making these proteins likely candidates for oxidation in acute inflammation. A8 is effectively oxidized in vitro by low amounts of reagent HOCl (molar ratios of Ͻϳ10), with 70 -80% conversion to dimer, and PMA activation of neutrophils generated an oxidative burst that efficiently oxidized exogenous A8 within 10 min, most likely via H 2 O 2 /MPO released by activated cells. (10). Moreover, disulfide-linked A8 homo-, but not A8/A9 heterodimer, is found in lung lavage fluid of mice at the beginning of the resolution phase of LPS-induced pulmonary injury (10), indicating oxidative modifications of A8 in inflammatory responses in vivo.
Oxidation of the single Cys 41 residue in A8 by Cu 2ϩ to the disulfide-linked homodimer was highly specific and negated bioactivity in vitro and in vivo (10). Positive chemotactic activity of a Cys 41 3 Ala 41 mutant (Ala 41 A8) confirmed that Cys 41 is not essential for function and implied that covalent dimerization may structurally modify accessibility of the chemotactic hinge domain. Therefore, oxidation-dependent dimerization may also be a physiologically significant regulatory mechanism controlling A8-provoked leukocyte recruitment (10). Moreover, the exquisite susceptibility of A8 to oxidation and the potentially large amounts present may protect against excessive tissue damage at sites of acute inflammation.
Here we describe the oxidation products of reagent HOCloxidized A8 using peptide mapping and mass spectrometry. Novel intra-and intermolecular sulfinamide bonds between Cys 41 and Lys residues were readily generated as well as conversion of Met to Met sulfoxide (Met(O)). Sulfinamide formation apparently occurs via sulfenic acid and/or sulfinyl chloride intermediates, which may decompose upon reaction with amines, yielding stable covalent bonds. Identical products were formed by HOCl generated enzymatically via the MPO/H 2 O 2 / Cl Ϫ system and from PMA-activated human neutrophils. The oxidized A8 monomer retained activity in chemotaxis assays in vitro.

EXPERIMENTAL PROCEDURES
General-Reagents and chemicals were analytical grade (Sigma, Bio-Rad), and solvents were HPLC grade (Mallinckrodt Chemical Works). Reagent hypochlorous acid (10 -13%) was from Aldrich. Recombinant A8/Ala 41 A8 was produced using the pGEX expression system as detailed (10,34). SDS/polyacrylamide gel electrophoresis/Western blotting were performed using a Mini Protean II apparatus (Bio-Rad) with 15% gels and a Tris/Tricene buffer system (35). Liquid chromatographic separations were performed using a non-metallic LC626 HPLC system (Waters, Bedford, MA) and monitored at A 214 nm and A 280 nm with a Waters 996 photodiode array detector or 490 UV-visible detector.
Oxidation of A8 by PMA-simulated Neutrophils-Neutrophils were isolated by standard procedures (10), washed twice, and resuspended in Dulbecco's PBS (Sigma). A8 (ϳ25 g, 2.5 nM) added to 10 7 cells/ml, activated with PMA (1 g/ml, Sigma), and incubated for 30 min at 37°C (Ϯ10 milliunits of MPO). Aliquots (500 l) removed 10 and 30 min after activation were centrifuged (10,000 g, 30 s), and supernatants were frozen immediately at Ϫ80°C. After thawing, oxidation products were separated using C4 RP HPLC (as above), and masses of oxidation products were determined using ESI-MS (as above). In some experiments sodium azide (50 M) was included to inhibit HOCl formation by MPO.

Site-directed Mutagenesis
The coding region of A8 was subcloned from a previously described construct (GST-CP10) (34) into the BamHI site of pBluescript (SK) vector (Stratagene, La Jolla, CA). The A8 insert was subcloned from pBluescript (SK) into the NotI/EcoRV sites of pOCUS-2 vector (Novagen, Madison, WI). The point mutations (Lys 34 and Lys 35 to Ala 34/35 ) were made using a modified version of the whole plasmid polymerase chain reaction technique described by Fisher and Pei (36), omitting the DpnI digestion step. Sequences of the reverse and forward primers used to generate the double point mutations were GAA GTC ATT CTT GTA GAG GGC ATG GTG ATT and GCG GCA ATG GTC ACT ACT GAG TGT CCT CAG. Both primers were phosphorylated using the T4 polynucleotide kinase (Roche Molecular Biochemicals) before polymerase chain reaction. After polymerase chain reaction and gel purification, the linearized plasmid was recircularized using T4 ligase (Promega, Madison, WI), then transformed in Escherichia coli. Plasmid DNA derived from individual colonies was bidirectionally sequenced to confirm that correct base substitutions had been made. The mutated A8-coding sequence was subcloned from pOCUS-2 vector into the BamHI site of pGEX-2T vector (Amrad-Pharmacia, Melbourne, Australia) and transformed into E. coli, and recombinant mutant protein was purified after isopropyl-1-thio-␤-D-galactopyranoside induction (34).

Mass Spectrometry
ESI-MS-Masses of proteins and peptides were determined using ESI. Spectra were acquired using a single quadrupole mass spectrometer equipped with an ESI source (MSD1100, Hewlett Packard, Palo Alto, CA). Samples (ϳ50 pmol, 10 l) were injected into water:acetonitrile (50:50), 1% acetic acid (10 l/min) using an LC1100 pump (Hewlett Packard) coupled directly to the electrospray source. Nitrogen was used as the nebulizer and drying gas (7.0 liter/min, 150°C). Sample droplets were ionized at a positive potential of ϳ4000 V and transferred to the mass analyzer with a fragmentor voltage (capillary to skimmer lens voltage) of 75 V. Spectra were acquired over the mass range m/z 200 -1500 in 0.5 s with unit resolution. Low energy collisional-induced dissociation (CID) MS/MS spectra were recorded using a triple quadrupole (TSQ 7000, Finnigan, San Jose, CA) where multiply charged (ϩ2, ϩ3, or ϩ4) precursor ions were selected with Q1, collisionally activated within rf-only Q2 (20 -100 eV, argon 1.5 milliotorr), and spectra were recorded with unit resolution using Q3 (m/z 50 -2550, 2 s) and accumulated into a single file for 4 -5 min. Ions were formed using an "in-house" nano-ESI device consisting of a bora-silicate glass capillary (50 ϫ 2.5 mm, exit inner diameter Ͻ50 m) containing peptide solutions (ϳ10 l, H 2 O/ CH 3 CN/CH 3 CO 2 H, 50:49:1). Electrical contact (ϳ1.5-2 kV) was maintained using a platinum electrode protruding into the liquid. The glass capillary was positioned 2-5 mm from the entrance to the heated capillary, which was at 150°C; stable ion currents were maintained with flow rates of Ͻ200 nl/min. MALDI-Protein or peptide solutions (ϳ25 pg/l) were mixed with matrix (1 l of sinapinic acid or 2,5-dihydroxybenzoic acid (Sigma) 10 mg/ml) and air-dried before analysis using a Voyager STR TOF mass spectrometer (Perseptive Biosystems, Framingham, MA). Spectra were acquired in linear mode, and positive ions were generated using an N 2 laser (337 nm, 3-ns pulse-width, 20-Hz repetition rate) and accelerated to 25 kV after an extraction delay of 100 -250 s. Typically, 25-50 spectra were averaged and calibrated externally using angiotensin I and insulin (oxidized) B chain (peptides) or insulin (oxidized) B chain and myoglobin (proteins).
Post source decay spectra (PSD) were also acquired using a Voyager STR mass spectrometer with the timed ion selector set to the precursor mass, a mirror ratio of 1.12; 11 segments with a 75% decrement ratio were acquired and "stitched" automatically using the instrument software (Data Explorer V5, Perseptive Biosystems). A different target position was chosen for each segment, and 100 -200 spectra were acquired and averaged. Spectra were calibrated using the metastable fragments of substance P or angiotensin-I (Sigma) and were generally Ͻϳ1.5 Da of their predicted value.

Chemotaxis Assay
A8 analogues (10 g) lyophilized (Speedvac, Savant, Farmingdale, NY) with glycerol (1 l) were stored at Ϫ80°C. Reconstitution to a 10 Ϫ6 M stock solution was with PBS (10 l) and RPMI 1640 (990 l) and serial dilutions were made in RPMI, bovine serum albumin (0.2%) immediately before use. Thioglycollate-elicited murine neutrophils were used as responding cells (15) and were suspended in HEPESbuffered saline solution, bovine serum albumin (0.2%) at 5 ϫ 10 6 /ml then incubated with calcein AM (5 M, Molecular Probes, Inc., Eugene, OR) for 15 min at 37°C in 5% CO 2 in air, washed twice with HEPESbuffered saline solution, and resuspended at 0.5 ϫ 10 6 /ml in RPMI, bovine serum albumin (0.2%). Assays were performed using MBA 96well chambers fitted with 5-m polycarbonate membranes (Neuro Probe Inc., Bethesda, MD). Proteins (10 Ϫ13 -10 Ϫ8 M) diluted in RPMI/ bovine serum albumin (0.2%, 410 l) were placed in the lower compartment, and cells (300 l) were placed in the upper compartment. To determine the effects of random migration, equivalent concentrations of protein were included in the upper chamber. The chamber was incubated at 37°C in 5% CO 2 in air for 90 min. Cells collected in the lower chamber were measured by fluorescence ( ex ϭ 485 nm, em ϭ 530 nm) using a multi-well plate reader (Cytofluor, Perseptive Biosystems), and numbers were extrapolated using a standard curve obtained using the fluorescence readings of a known number of labeled cells. Complement C5a (10 Ϫ9 M) was used as positive control in all experiments. Data from at least three experiments were analyzed using Student's t test.

RESULTS AND DISCUSSION
Hypochlorite (OCl Ϫ ) is the major oxidant produced by neutrophils in inflammatory responses, and murine A8 is highly susceptible to oxidation by reagent HOCl (10). Low concentrations of hypochlorite (Ͻ40 M) convert A8 to a modified monomer (10,354, ϩ46 Da), corresponding to the addition of 3 oxygens and loss of dihydrogen and to oxidized dimer (20,707, ϩ93 Da), indicating oxidation of susceptible amino acids (Cys/Met) (10). SDS-polyacrylamide gel electrophoresis separation with silver staining showed that when A8 was treated with as little as 10 M HOCl, ϳ20% was converted to dimer and yields increased to 70 -80% with 40 M HOCl, whereas levels Ͼ100 M caused loss of detectable protein, possibly due to aggregation. The resistance of Ala 41 A8 to HOCl-mediated dimerization at all HOCl concentrations confirmed the role of Cys 41 .
Reagent or MPO-generated HOCl (28) form identical reaction products in proteins with reactive aldehydes from ␣-amino acids (37), and Cys and Met, followed by amines (e.g. Lys, forming chloramines), are substrates (30,38,39). Thus, products generated by reagent HOCl in vitro are likely to reflect those produced by HOCl generated by activated phagocytes.
The minor peak (Fig. 1A, peak 2), eluting as a partially resolved shoulder immediately preceding peak 3, had an m/z of 10,388 (i.e. ϩ80, or the addition of 5 oxygens) and corresponds to fully oxidized A8 (i.e. Met 36/73 to Met 36/73 (O) and Cys 41 to cysteic acid). MS of Met/Cys-containing Asp-N digest peptides confirmed the Met(O) and cysteic acid products (not shown).
The mass addition (m/z ϩ46) of the major A8 oxidation product (Fig. 1A, peak 3) was not totally accounted for by Met oxidation, suggesting that Cys 41 was modified by the addition of 14 Da. This would numerically correspond to the addition of a single oxygen (m/z ϩ16) to Cys to form Cys sulfenic acid (Cys(O)) followed by loss of dihydrogen (m/z Ϫ2). Mechanistically, HOCl oxidation of Cys to cysteic acid (CysSO 3 H) proceeds stepwise via Cys sulfenic and Cys sulfinic acid (CysSO 2 H) intermediates (40). Sulfenic acids are transient intermediates that readily undergo further oxidation or substitution reactions but may be stabilized within proteins by hydrogen bonding to carbonyl or amino groups (40). Protein-containing Cys(O) are proposed as important biochemical intermediates located at active site Cys residues and possibly responsible for oxidative inactivation of enzymes such as papain and glyceraldehyde-3phosphate dehydrogenase (40). Importantly, redox regulation of the transcription factors Fos, Jun, and OxyR and the reversible inactivation of protein-tyrosine phosphatase 1B and CD45 may also involve Cys to Cys(O) conversion (40 -42). Intermediates of bacterial flavoprotein thioredoxin reductase may also contain Cys(O) (43). Because of their instability, Cys(O)-protein intermediates have been difficult to isolate. Cys(O) in the enterococcal flavoprotein NADH peroxidase was identified and characterized using x-ray crystallography under cryogenic conditions (40,41), and the Cys(O) adduct of alkyl hydroperoxidase reductase was identified after prior trapping with the electrophilic reagent 12-(N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4yl))Cl (44).
Sulfenic acids undergo substitution reactions with nucleophilic reagents such as dimedone (44,45) to form thiol adducts that would yield a calculated mass increase of ϩ138 Da. After incubation of the A8/HOCl reaction mixture with dimedone and C4 RP-HPLC separation of the products, two additional components with masses of m/z 10,477 and 10,444 (Fig. 1B, peaks 3a and 3b) indicated dimedone adducts. Asp-N peptide mapping confirmed dimedone substitution on A8 32-57 and low energy CID of the [M ϩ 3H ϩ ] 3ϩ ion showed fragmentations consistent with the addition to Cys 41 (not shown). The observed mass difference (m/z ϳ32) found between peaks 3a and 3b (Fig.  1B) was due to incomplete Met oxidation. Interestingly, A8dimedone adducts were evident only if excess dimedone was added within ϳ20 s of initiation of HOCl oxidation, suggesting that conversion of A8 sulfenic acid to the stable products isolated here (Fig. 1A) occurred almost immediately. Early publications suggest that protein sulfenic acids may undergo nucleophilic substitution with amines (e.g. benzylamine) to yield stable covalent bonds (45), although none have been isolated or rigorously characterized. Our results suggest that A8 oxidation products form after reaction of specific amino acids with a Cys(O) intermediate.
MALDI Reflectron TOF Peptide Mapping of A8 Oxidation Products-Definitive characterization of the mass additions of all A8 HOCl oxidation products was facilitated by peptide mapping-MALDI reflection TOF MS, which allows mass accuracies of Ͻϳ0.2 Da and resolution of isotopic envelopes in peptides of m/z Ͻϳ7000, and tandem MS (46). The complete peptide mass profiles of oxidized A8 adducts isolated from C4 RP-HPLC (Fig.  1A) were determined after Asp-N digestion (without separation of peptides by RP-HPLC). Comparison of experimental and predicted masses of covalently linked oxidized Asp-N peptides (Fig. 2) confirmed the proposed oxidative additions and mass assignments determined using ESI-MS of the full-length proteins (Fig. 1).
The spectrum of the A8 Asp-N digest ( Fig. 2A) gave the expected digest peptides (47), and experimental masses of the most intense ions (m/z 615. 16 ) also differed by m/z ϳ0.2, and the expanded mass scale showed resolved isotopic peaks ( Fig. 2A, inset, resolution M/⌬M ϳ5000, 50% peak height), which also agreed well with the calculated isotopic pattern (not shown). Reflectron TOF-MS of Asp-N digest products confirmed the identity of fully oxidized A8 (Fig. 1A, peak 2 The major oxidation product (Fig. 1A, peak 3, m/z 10,354) yielded two peptides with masses distinct from those derived from native A8 (Fig. 2C). 2) confirmed the unusual mass addition found in the full-length protein and substantiated its location within A8  . The mass of the minor early eluting oxidation product (Fig. 1A, peak 1) was identical to that of the major component above, suggesting an alternative isomeric reaction products form. Reflectron TOF-MS of Asp-N digest products indicated intense ions with m/z 1457.61, 2287.98, 2448.30 (Fig. 2D), identical to the protonated peptides A8 Ϫ2-12 , A8 13-31 , and Met(O)-A8  . No peptide corresponding to A8 32-57 was evident, although there was an additional ion at m/z 3802.7562. Calculated masses of possible A8 Asp-N products indicated that this would correspond to the average mass of A8 32-57 if it were covalently linked to A8 84 -88 (with the addition of m/z 30). The isotopic distribution pattern of the protonated peptide showed an almost identical profile to the calculated isotopic pattern for A8 32-57/84 -88 containing 2 oxygens (Ϫ2 hydrogens; formula: C 165 H 259 N 44 O 55 S 2 ) (not shown). The monoisotopic masses differed by Ͻ0.2 Da, confirming that a minor component formed after HOCl oxidation of A8 contained a covalent bond between A8 32-57 and A8 84 -88 , with the addition of m/z 14.
One oxidized component (peak 4 , Fig. 1A) had a numerical mass corresponding to a dimer between 2 oxidized monomeric chains. This was 28 Da greater than predicted for the disulfide- , and this exhibited a very similar monoisotopic mass and isotopic distribution pattern compared with those predicted (inset, Fig. 2E). Reduction with dithiothreitol (1 mM, 37°C, 30 min) did not alter the masses of A8 dimers or oxidized monomers (not shown), indicating that this unusual cross-linking is resistant to conventional reductants. Taken together, the dimer most likely contains novel intramolecular and/or intermolecular cross-links between two A8 chains.
A Novel Covalent Cys Modification in Oxidized A8 -Each peptide covalently linked to oxidized Cys 41 contained a free amine located on the ⑀-amine of Lys (i.e. Lys 87 (Fig. 1, peak 1), Lys 34/35 (peak 3), and Lys 6 (peak 4). Amines may react with Cys(O) to form stable products containing covalent bonds such as that found after reaction of the sulfenic acid form of papain with benzylamine (45). We propose that hypochlorite rapidly oxidizes Cys 41 to Cys 41 (O), which is partially stabilized, possibly by hydrogen bonding with amines on neighboring Lys residues. The sulfenic acid may then undergo nucleophilc substitution by the ⑀-amine of Lys, followed by loss of dihydrogen to yield sulfinamides. Cys sulfinyl chloride may be an intermediate. Fig. 3 summarizes the proposed major HOCl oxidation products of A8, and Scheme 1 shows the possible mechanism of sulfinamide cross-linking. All major products contained Met(O). Based on the relative levels of oxidation products (Fig.  1A), the Lys residues most structurally favorable for intermolecular cross-linking would be Lys 34 or Lys 35 and then Lys 87 or Lys 6 . HOCl oxidation of glutathione (GSH) may occur via a similar process, oxidized dimeric GSH and monomeric GSH containing a cyclic sulfonamide (with two oxygens on Cys) were identified (50). Winterbourn and Brennan (50) propose the initial formation of GSH-sulfenyl chloride followed by oxidation  (Fig. 1A, peaks 2, 3, 1, and 4) were digested with Asp-N and analyzed directly by MALDI after the addition of matrix (B, C, D, and E, respectively). Masses of the most intense ions and identities of expected digest peptides are indicated. Peptides with spectra characteristic of Cys 41 / Lys x sulfinamides are also given as expanded mass axes showing isotopic resolution and monoisotopic mass (insets). The spectrum of the fully oxidized protein (Fig. 1, peak 2) contains an ion at m/z 3222.2029, corresponding to A8 32-57 , having five additional oxygens (B). The spectrum of the major oxidation product (Fig. 1, peak 3)   to GSH-sulfonyl chloride and then either cyclization with the free N-terminal amine (to yield a cyclic sulfonamide) or the addition of GSH to form a partially oxidized dimer. No sulfonamide bonds between Cys 41 and Lys residues were identified in oxidized A8. Differences in the oxidation products of proteins and GSH may be due to stabilization of initially formed Cys(O) by hydrogen bonding to Lys residues, the reactivity of respective Cys residues, the amount of oxidant used, and/or more favorable structural/spatial arrangements in proteins compared with the relative distances of amino acid side chains within GSH. Interestingly, HOCl oxidation of the peptide A8 32-57 (containing Lys 34/35 and Cys 41 ) with a similar molar ratio of reagents yielded a single product that corresponded to fully oxidized peptide containing Met(O) and cysteic acid (not shown) rather than the sulfinamide cross-linked products, suggesting that the full-length protein is in the most favorable structural orientation for sulfinamide formation. The threedimensional structures of related S100 proteins (e.g. human S100A8, S100B) indicate that residues 32-40 are located with an ␣-helix (helix II), and the side chains of Lys 34/35 and Cys 41 are relatively closely spaced (51,52). A helical wheel representation of A8 also supports an ␣-helical structure with Lys 34/35 and Cys 41 residues on adjacent helices and favorably positioned to allow cyclization and intermolecular sulfinamide cross-linking. Although the predominant sulfinamide bond was within monomers and even though A8 forms non-covalent dimers in solution (53), the products containing intramolecular sulfinamides between Lys 87 /Cys 41 and Lys 6 /Cys 41 (Fig. 3) indicate some selective cross-linking between Cys 41 (O) on one chain with available Lys residues on the second chain.
Tandem MS Identification of Covalently Linked Amino Acids in the Major A8 Oxidation Product-Low energy CID MS/MS spectra of the Asp-N digest peptide A8 32-57 of oxidized and native A8 (Fig. 4A) isolated by RP-HPLC showed characteristic fragmentation patterns (y 4 Ј to y 16 ЈЈ) confirming that residues 43-57 were not modified (data not shown). Peptides containing fewer amino acids were required for definitive MS/MS characterization. Fig. 4 summarizes the masses of peptides isolated by C18 RP-HPLC after Lys-C, Glu-C, and pepsin digestion of native (Fig. 4B) and oxidized A8   (Fig. 4C). The peptides generally contained the usual sequence-specific b-or y-type MS/MS fragmentations, but several also had characteristic fragmentations that could be attributed to intramolecular sulfinamide bonds (Fig. 4). Peptides with masses of m/z 409 and 2640 and corresponding to the predicted masses of A8 32-34 and A8 36 -57 were isolated after complete digestion of A8 32-57 with Lys-C (Fig. 4B). In contrast, three peptides of masses m/z 409, 2798, and 3206 and corresponding to oxidized A8 32-34 , A8  , and A8 32-57 ϩ H 2 O, were generated from oxidized A8 32-57 ( Fig.  4C and 5A). Those with masses of m/z 409 and 2798 were expected products of oxidized A8 32-57 generated from digestion at Lys 34 , confirming that amino acids 32-34 were not modified. The peptide with mass m/z 3206 was m/z ϩ18 more than oxidized A8  , and the mass increase corresponded to the addition of H 2 O. These results suggest that an unusual intramolecular covalent link (sulfinamide) within this peptide prevented dissolution of peptide DFKK after digestion at Lys 35 and indicates that Lys-C cleaved between residues Lys 34 -Lys 35 and Lys 35 -Met(O) 36 to generate two peptides (Fig. 5A). Taken together, covalent sulfinamide bonds between Cys 41 and Lys 34 or Lys 35 were indicated, and oxidized A8 32-57 contained a mixture of isomers, A8 32-57 Cys 41 /Lys 34 -sulfinamide and A8  Cys 41 /Lys 35 -sulfinamide, which were not initially separated by C18 RP-HPLC of the Asp-N digest.
MALDI PSD Mass Spectrometry-Although PSD tandem MS suffers from low resolution precursor ion selection (M/⌬M ϳ80) when using the timed ion selector of the Voyager mass spectrometer, useful structural information can be obtained from the fragmentations obtained from HPLC-fractionated peptides, where the homogeneity of the precursor ion can be assured (54). The PSD spectra of protonated oxidized A8 32-57 ϩ H 2 O and A8  showed characteristic fragmentations that confirmed the most likely covalently linked residues as Cys 41 (O) and Lys 34 or Lys 35 forming sulfinamide bonds ( Fig. 5B and data not shown). Four major fragments were evident in the PSD spectra of the [M ϩ H] ϩ ion of oxidized A8 32-57 ϩ H 2 O) (Fig. 5B). The ion at m/z 2622.8 corresponds to loss of m/z 584, which could be attributed to the loss of DFKK( ⑀ NHSOH) (calculated mass, m/z 585) and formation of dehydo-alanine at the Cys residue. A proposed fragmentation mechanism is outlined (Fig. 5C). Processes whereby protonated, oxidized, and substituted Cys residues preferentially undergo elimination reactions to form protonated peptides containing dehydro-alanine and substituted sulfenic acids have recently been described (55). Furthermore, the ion at m/z 2559.1 corresponds to the loss of methanesulfenic acid (m/z 64) from the ion at m/z 2622.8, a characteristic fragmentation of Met(O) with numerous examples reported (56). Loss of methanesulfenic acid from the precursor ion is also evident, forming the ion at 3141 (Fig. 5C). Another fragmentation producing an ion at 1974.6, and corresponding to yЈЈ 16  . Simple fragmentation of the sulfinamide bond forming two protonoted products accounts for the observed spectrum (data not shown).
The PSD spectra of the [M ϩ H] ϩ ion of oxidized A8 35-57 contained one major ion at m/z 1974.6, which corresponded to yЈЈ 16 (Fig. 6A and B). Pepsin digestion of A8 caused loss of the very basic Arg residue near the C terminus, shifting the fragmentation pattern from predominantly y-to b-type. The almost complete series of b ions (b 12 -b 3 ) in the MS/MS spectrum of protonated A8 32-44 indicated loss of individual amino acids from the C terminus and retention of charge at the N terminus (Fig. 6A). Predominant fragmentations around Cys 41 (b 10 and b 9 ) were obvious. Except for the addition of 16 Da to fragments b Ͼ 4 (corresponds to conversion of Met 36 to Met 36 (O)) and an additional 14 Da for fragments b Ͼ 9 (e.g. b 10 m/z 1214.3) (Fig. 6B), spectra of protonated A8 32-44 and oxidized A8 32-44 were similar. Additional fragments attributed to  (Fig. 6B), confirming formation of a Cys sulfinyl derivative. Similar spectra were obtained after PSD analysis of the same peptic peptides, confirming the location of the oxidized amino acids and an intramolecular sulfinamide bond between Lys 34/35 and oxidized Cys 41 (data not shown) and accounting for an overall increase in mass of 46 Da. The intense ion at m/z 1231 did not correspond to the calculated mass of any expected ion, suggesting that this fragmentation may be specific for the sulfinamide or represent a rearrangement ion. A possible fragmentation mechanism may involve sequential losses of C-terminal amino acids to form a b 10 ϩ H 2 O ion, which has a calculated mass of m/z 1231.4, almost identical to the observed experimental mass. Although uncommon, this type of process is favored in peptides containing basic N-terminal amino acids (57,58). Additional experiments using a quadrupole ion trap and MS 3 may clarify the identity of this unusual fragment ion. S100A8 Oxidation Products from Cellular or H 2 O 2 /MPOgenerated HOCl-Products with almost identical HPLC profiles to those produced by reagent HOCl (Fig. 7B) were isolated after oxidation by HOCl generated by MPO/H 2 O 2 /Cl Ϫ (Fig. 7C). In contrast, H 2 O 2 oxidation of A8 for 10 min yielded only low levels of disulfide-bonded dimer (mass m/z 20614, Fig. 7D), which increased after prolonged incubation (T Ͼ 60 min, not shown), with little evidence of Met oxidation. These results indicate preferential Cys oxidation by H 2 O 2 , probably via thiyl radicals (59). The masses of the major reagent HOCl oxidation products were m/z 10354 and 20707 (Fig. 7B) and m/z 10354 and 20680/20707 when MPO/H 2 O 2 /Cl Ϫ was used (Fig. 7C), indicating production of some disulfide-bonded A8 dimer containing Met (O) (m/z calculated, 20,678). Products with similar C4 retention times and identical masses were isolated after stimulation of human neutrophils with PMA containing exogenous A8 (Fig. 7E), although the chromatograms and mass spectra were complicated by secreted human neutrophil proteins.
Chemotactic Activity of Oxidized A8 Monomer-Substitution of Cys 41 in A8 does not affect its function (10) and the Lys-Ala mutant generated for the studies presented here (Ala 34/35 A8) also retained full activity (not shown). A8 and Ala 41 A8 (10 Ϫ11 -10 Ϫ8 M) induced dose-dependent migration of neutrophils (Fig.  8), with optimal activity at 10 Ϫ10 M (p Ͻ 0.01 compared with control). The major HOCl oxidation product containing Cys 41 / Lys 34/35 sulfinamide (oxA8) exhibited responses comparable with Ala 41 A8 (p Ͻ 0.01), and equal amounts in the upper and lower wells of the chemotaxis chambers abolished migration (p Ͻ 0.01 relative to maximal chemotaxis at 10 Ϫ9 and 10 Ϫ10 M Ala 41 A8 in the lower chamber), confirming that activity was not due to random migration. The positive activity of the monomeric sulfinamide contrasted markedly to the inactivation caused by copper oxidation, which generates covalent S-S homodimers (10). Difficulties in separating sufficient pure sulfinamide-containing dimer has not yet allowed its activity to be assessed, The relative amounts of modified A8 monomers and dimers formed were dependent on the concentration of HOCl used and reaction time (not shown), and aggregation predominated with molar ratios Ͼϳ10 fold (not shown), suggesting a two-step process. Oxidation-dependent modifications in A8 may represent a physiologically significant regulatory mechanism con-FIG. 7. Comparison of C4 RP-HPLC chromatograms after incubation of S100A8 with various oxidants. A, unmodified A8. Similar chromatograms were obtained after the oxidation with reagent HOCl (molar ratio protein:oxidant ϳ1:5) (B) or myeloperoxidase/H 2 O 2 / Cl Ϫ (molar ratio protein:H 2 O 2 ϳ1:5) (C) for 10 min. The reaction of H 2 O 2 without MPO (D) (molar ratio protein:H 2 O 2 ϳ1:5) converted a small amount of monomer to disulfide-linked dimer in 10 min. PMA-activated human PMN also converted exogenous A8 into oxidized forms, although the chromatogram was complicated by secreted PMN proteins. trolling A8-provoked leukocyte recruitment (10). In conditions of low HOCl production, sulfinamide bond formation may stabilize the monomeric chemotactic form and favor continued leukocyte influx, whereas higher amounts of HOCl could generate intra-chain cross-linked inactive products, resulting in decreased inflammation. Importantly, the sensitivity of A8 to oxidation may represent a novel means of protecting host tissue from excessive oxidative damage when high levels of HOCl are generated. A8 constitutes ϳ20% of the cytoplasmic protein of neutrophils, and large amounts of extracellular murine A8 are found in acute inflammatory sites, including bacterial infection (19) and lung alveolitis (18). High levels of the human protein are found in serum from patients with numerous acute and chronic inflammatory conditions (5). Furthermore, A8 gene transcription and protein secretion by macrophages activated by endotoxin or interferon ␥ is markedly amplified by interleukin 10 (IL10), and IL10 from LPS-activated cells is partially responsible for the LPS-provoked response. Interleukin 10 acts via a prostaglandin E 2 -cAMP pathway, supporting the notion that A8 may be involved in protection/resolution of cell-mediated immune responses (60).
Inside cells, S100 proteins occur mainly as non-covalent homo-/heterodimers with reduced sulfhydryl groups (32), although we recently identified A8 S-S homodimers exclusively in blood neutrophils and in a heterogeneous neutrophil population at inflammatory sites (61), suggesting that this protein may contribute to maintenance of a reducing environment in these cells. In the case of S100B, conversion of the monomer to the disulfide dimer (62,63) or an intramolecular S-S bonded form (9) enhances its neurotrophic and mitogenic activities. S100A2 in keratinocytes also undergoes intramolecular crosslinking under oxidative stress and is proposed to protect against carcinogens (11). In view of the sensitivity of Cys residues in A8 to oxidation by physiological oxidants such as HOCl and of the potentially important functional role of these residues in defining target protein-binding sites in S100 proteins (64,65), we suggest that Cys modification may represent a common S100 regulatory mechanism.