Conformational mapping of the N-terminal peptide of HIV-1 gp41 in membrane environments using C-enhanced Fourier transform infrared spectroscopy

The N-terminal domain of HIV-1 glycoprotein 41?000 (FP; residues 1-23; AVGIGALFLGFLGAAGSTMGARSCONH₂) participates in fusion processes underlying virus-cell infection. Here, we use physical techniques to study the secondary conformation of synthetic FP in aqueous, structure-promoting, lipid and biomembrane environments. Circular dichroism and conventional, ¹²C-Fourier transform infrared (FTIR) spectroscopy indicated the following α-helical levels for FP in 1-palmitoyl-2-oleoylphosphatidylglycerol (POPG) liposomes∼hexafluoroisopropanol (HFIP)>trifluoroethanol (TFE)>phosphate-buffered saline (PBS). ¹²C-FTIR spectra also showed disordered FP structures in these environments, along with substantial β-structures for FP in TFE or PBS. In further experiments designed to map secondary conformations to specific residues, isotope-enhanced FTIR spectroscopy was performed using a suite of FP peptides labeled with ¹³C-carbonyl at multiple sites. Combining these ¹³C-enhanced FTIR results with molecular simulations indicated the following model for FP in HFIP: α-helix (residues 3-16) and random and β-structures (residues 1-2 and residues 17-23). Additional ¹³C-FTIR analysis indicated a similar conformation for FP in POPG at low peptide loading, except that the α-helix extends over residues 1-16. At low peptide loading in either human erythrocyte ghosts or lipid extracts from ghosts, ¹³C-FTIR spectroscopy showed α-helical conformations for the central core of FP (residues 5-15); on the other hand, at high peptide loading in ghosts or lipid extracts, the central core of FP assumed an antiparallel β-structure. FP at low loading in ghosts probably inserts deeply as an α-helix into the hydrophobic membrane bilayer, while at higher loading FP primarily associates with ghosts as an aqueous-accessible, β-sheet. In future studies, ¹³C-FTIR spectroscopy may yield residue-specific conformations for other membrane-bound proteins or peptides, which have been difficult to analyze with more standard methodologies.     

Spectroscopic Determination of Lysozyme Conformational Changes in the Presence of Trehalose and Guanidine

The bioprotective action of the disaccharide trehalose has been studied against the well-known denaturating agent, guanidine hydrochloride. The results indicated a direct influence of trehalose on both enzymatic activity and conformational changes of lysozyme, as shown by the decrease of the inactivation rate constant of about 1.48-fold and the loss of α-helix structure of lysozyme. In addition, ESI–MS hydrogen–deuterium (H/D) exchange experiments allowed us to correlate the structural and dynamic features of the protein in the presence of the two additives, highlighting as trehalose remarkably influenced this exchange by decreasing local protein environment changes and solvent accessibility to the amide peptide backbone, as further evidenced by circular dichroism and ¹H NMR measurements.     

Conformational mapping of the N-terminal peptide of HIV-1 gp41 in membrane environments using (13)C-enhanced Fourier transform infrared spectroscopy

The N-terminal domain of HIV-1 glycoprotein 41000 (FP; residues 1--23; AVGIGALFLGFLGAAGSTMGARSCONH(2)) participates in fusion processes underlying virus--cell infection. Here, we use physical techniques to study the secondary conformation of synthetic FP in aqueous, structure-promoting, lipid and biomembrane environments. Circular dichroism and conventional, (12)C-Fourier transform infrared (FTIR) spectroscopy indicated the following alpha-helical levels for FP in 1-palmitoyl-2-oleoylphosphatidylglycerol (POPG) liposomes-hexafluoroisopropanol (HFIP)>trifluoroethanol (TFE)>phosphate-buffered saline (PBS). (12)C-FTIR spectra also showed disordered FP structures in these environments, along with substantial beta-structures for FP in TFE or PBS. In further experiments designed to map secondary conformations to specific residues, isotope-enhanced FTIR spectroscopy was performed using a suite of FP peptides labeled with (13)C-carbonyl at multiple sites. Combining these (13)C-enhanced FTIR results with molecular simulations indicated the following model for FP in HFIP: alpha-helix (residues 3-16) and random and beta-structures (residues 1-2 and residues 17-23). Additional (13)C-FTIR analysis indicated a similar conformation for FP in POPG at low peptide loading, except that the alpha-helix extends over residues 1-16. At low peptide loading in either human erythrocyte ghosts or lipid extracts from ghosts, (13)C-FTIR spectroscopy showed alpha-helical conformations for the central core of FP (residues 5-15); on the other hand, at high peptide loading in ghosts or lipid extracts, the central core of FP assumed an antiparallel beta-structure. FP at low loading in ghosts probably inserts deeply as an alpha-helix into the hydrophobic membrane bilayer, while at higher loading FP primarily associates with ghosts as an aqueous-accessible, beta-sheet. In future studies, (13)C-FTIR spectroscopy may yield residue-specific conformations for other membrane-bound proteins or peptides, which have been difficult to analyze with more standard methodologies.     

Comparison of experimental and theoretical data on hydrogen-deuterium exchange for ten globular proteins

The number of protons available for hydrogen-deuterium exchange was predicted for ten globular proteins using a method described elsewhere by the authors. The average number of protons replaced by deuterium was also determined by mass spectrometry of the intact proteins in their native conformations. Based on these data, we find that two models proposed earlier agree with each other in estimation of the number of protons replaced by deuterium. Using a model with a probability scale for hydrogen bond formation, we estimated a number of protons replaced by deuterium that is close to the experimental data for long-term incubation in D₂O (24 h). Using a model based on estimations with a scale of the expected number of contacts in globular proteins there is better agreement with the experimental data obtained for a short period of incubation in D₂O (15 min). Therefore, the former model determines weakly fluctuating parts of a protein that are in contact with solvent only for a small fraction of the time. The latter model (based on the scale of expected number of contacts) predicts either flexible parts of a protein chain exposed to interactions with solvent or disordered parts of the protein.     

Mass spectrometric approaches using electrospray ionization charge states and hydrogen-deuterium exchange for determining protein structures and their conformational changes

Electrospray ionization (ESI) mass spectrometry (MS) is a powerful analytical tool for elucidating structural details of proteins in solution especially when coupled with amide hydrogen/deuterium (H/D) exchange analysis. ESI charge-state distributions and the envelopes of charges they form from proteins can provide an abundance of information on solution conformations that is not readily available through other biophysical techniques such as near ultraviolet circular dichroism (CD) and tryptophan fluorescence. The most compelling reason for the use of ESI-MS over nuclear magnetic resonance (NMR) for measuring H/D after exchange is that larger proteins and lesser amounts of samples can be studied. In addition, MS can provide structural details on transient or folding intermediates that may not be accessible by CD, fluorescence, and NMR because these techniques measure the average properties of large populations of proteins in solution. Correlations between measured H/D and calculated parameters that are often available from crystallographic data can be used to extend the range of structural details obtained on proteins. Molecular dynamics and energy minimization by simulation techniques such as assisted model building with energy refinement (AMBER) force field can be very useful in providing structural models of proteins that rationalize the experimental H/D exchange results. Charge-state envelopes and H/D exchange information from ESI-MS data used complementarily with NMR and CD data provides the most powerful approach available to understanding the structures and dynamics of proteins in solution.     

Metabolic profiling of saponins in Medicago sativa and Medicago truncatula using HPLC coupled to an electrospray ion-trap mass spectrometer

Triterpene saponins isolated from Medicago sativa (alfalfa) and Medicago truncatula roots were separated, profiled and identified using an optimized, reversed-phase HPLC with on-line photodiode array detection and electrospray ionization mass spectrometry method (HPLC/PDA/ESI/MS). ESI source polarity and solvent conditions were compared. The effects of these parameters on mass spectral attributes were determined. Ion structures were confirmed using tandem mass spectrometry (MS/MS). Fifteen saponins were identified in alfalfa (cultivars Apollo, Radius, and Kleszczewska) based upon negative-ion HPLC/PDA/ESI/MS, HPLC/PDA/ESI/MS/MS and literature data. In addition, the identification of two new malonated saponins in alfalfa are proposed. Negative-ion HPLC/PDA/ESI/MS and HPLC/PDA/ESI/MS/MS spectra were utilized along with HPLC retention times to profile and identify 27 saponins in M. truncatula (cultivar Jemalong, A17). M. truncatula yielded a much more complex mixture of saponins than observed for alfalfa. The authors are not aware of any previous reports identifying saponin glycosides in M. truncatula.     

Deuterium exchange of alpha-helices and beta-sheets as monitored by electrospray ionization mass spectrometry.

Deuterium exchange was monitored by electrospray ionization mass spectrometry (ESI-MS) to study the slowly exchanging (hydrogen bonded) peptide hydrogens of several alpha-helical peptides and beta-sheet proteins. Polypeptides were synthetically engineered to have mainly disordered, alpha-helical, or beta-sheet structure. For 3 isomeric 31-residue alpha-helical peptides, the number of slowly exchanging hydrogens as measured by ESI-MS in 50% CF3CD2OD (pD 9.5) provided estimates of their alpha-helicities (26%, 40%, 94%) that agreed well with the values (17%, 34%, 98%) measured by circular dichroic spectroscopy in the same nondeuterated solvent. For 3 betabellins containing a pair of beta-sheets and a related disordered peptide, their order of structural stability (12D > 12S > 14D > 14S) shown by their deuterium exchange rates in 10% CD3OD/0.5% CD3CO2D (pD 3.8) as measured by ESI-MS was the same as their order of structural stability to unfolding with increasing temperature or guanidinium chloride concentration as measured by circular dichroic spectroscopy in water. Compared to monitoring deuterium exchange by proton NMR spectrometry, monitoring deuterium exchange by ESI-MS requires much less sample (1-50 micrograms), much shorter analysis time (10-90 min), and no chemical quenching of the exchange reaction.     

13C-nmr chemical shift and conformation of L-alanine residues incorporated into poly(β-benzyl L-aspartate) in the solid state

¹³C-nmr spectra of poly(β-benzyl L-aspartate) containing ¹³C-enriched [3-¹³C]L -alanine residues in the solid state were recorded by the cross polarization–magic angle spinning method, in order to elucidate the conformation-dependent ¹³C chemical shifts of L -alanine residues taking various conformations such as the antiparallel β-sheet, the right-handed α-helix, the left-handed α-helix, and the left-handed ω-helix forms obtained by appropriate treatment. The latter two conformations for L -alanine residues are achieved when L -alanine residues are incorporated into poly(β-benzyl L -aspartate). We found that the alanine C_β carbon show significant ¹³C chemical shift displacement depending on conformational change, and gave the ¹³C chemical shift values at about 17 ppm for the left-handed ω-helix, 14 ppm for the left-handed α-helix, 15.5 ppm for the right-handed α-helix, and 21.0 ppm for the antiparallel β-sheet relative to tetramethylsilane.     

Remodeling of HIV-1 Nef Structure by Src-Family Kinase Binding

The HIV-1 accessory protein Nef controls multiple aspects of the viral life cycle and host immune response, making it an attractive therapeutic target. Previous X-ray crystal structures of Nef in complex with key host cell binding partners have shed light on protein–protein interactions critical to Nef function. Crystal structures of Nef in complex with either the SH3 or tandem SH3–SH2 domains of Src-family kinases reveal distinct dimer conformations of Nef. However, the existence of these Nef dimer complexes in solution has not been established. Here we used hydrogen exchange mass spectrometry (HX MS) to compare the solution conformation of Nef alone and in complexes with the SH3 or the SH3–SH2 domains of the Src-family kinase Hck. HX MS revealed that interaction with the Hck SH3 or tandem SH3–SH2 domains induces protection of the Nef αB-helix from deuterium uptake, consistent with a role for αB in dimer formation. HX MS analysis of a Nef mutant (position Asp123, a site buried in the Nef:SH3 dimer but surface exposed in the Nef:SH3–SH2 complex), showed a Hck-induced conformational change in Nef relative to wild-type Nef. These results support a model in which Src-family kinase binding induces conformational changes in Nef to expose residues critical for interaction with the μ1 subunit of adaptor protein 1 and the major histocompatibility complex-1 tail, and subsequent major histocompatibility complex-1 downregulation and immune escape of HIV-infected cells required for functional interactions with downstream binding partners.     

The conformational characteristics in solution of the synthetic cyclic hexapeptide 5L-ala-D-ala

An attempt to elucidate the solution conformation(s) of the synthetic cyclic hexapeptide 5L -ala·D-ala is described. Nuclear magnetic resonance (nmr) spectra are recorded for the purpose of measuring the vicinal coupling constant between the amide and α-protons in each residue and to observe the deuterium exchange rate and temperature dependence of the chemical shift of each amide proton. Low-energy cyclic conformations, whose individual residues are in conformations consistent with the observed amide to α-proton coupling constant, are searched for in an approximate theoretical treatment. The two lowest energy, all trans peptide bond conformations generated are distinguishable by the presence or absence of a single intramolecular hydrogen bond. The observed temperature independence of the chemical shift of one of the amide protons is consistent with the presence of a single intramolecular hydrogen bond, while the observation of similar deuterium exchange rates for each of the amide protons indicates their comparable availability to solvent. Consequently, it is concluded that 5L -ala·D-ala is in rapid equilibrium between conformations with and without a single internal hydrogen bond and possesses considerable conformational flexibility in solution.     

Characterization of peptides resulting from digestion of human skin elastin with elastase

Several pathological disorders are associated with abnormalities in elastic fibers, which are mainly composed of elastin. Understanding the biochemical basis of such disorders requires information about the primary structure of elastin. Since the acquisition of structural information for elastin is hampered by its extreme insolubility in water or any organic solvent, in this study, human skin elastin was digested with elastase to produce water-soluble peptides. Tandem mass spectrometry (MS/MS) experiments were performed using conventional electrospray ionization (ESI) and nano-ESI techniques coupled with ion trap and quadrupole time-of-flight (qTOF) mass analyzers, respectively. The peptides were identified from the fragment spectra using database searching and/or de novo sequencing. The cleavage sites of the enzyme and, for the first time, the extent and location of proline hydroxylation in human skin elastin were determined. A total of 117 peptides were identified with sequence coverage of 58.8%. It has been observed that 25% of proline residues in the sequenced region are hydroxylated. Elastase cleaves predominantly at the C-terminals of the amino acids Gly, Val, Leu, Ala, and Ile, and to a lesser extent at Phe, Pro, Glu, and Arg. Our results confirm a previous report that human skin elastin lacks amino acid sequences expressed by exon 26A.     

Proline-induced constraints in alpha-helices

The disrupting effect of a prolyl residue on an α-helix has been analyzed by means of conformational energy computations. In the preferred, nearly α-helical conformations of Ac-Ala₄-Pro-NHMe and of Ac-Ala₇-Pro-Ala₇-NHMe, only the residue preceding Pro is not α-helical, while all other residues can occur in the α-helical A conformation; i.e., it is sufficient to introduce a conformational change of only one residue in order to accommodate proline in a distorted α-helix. Other low-energy conformations exist in which the conformational state of three residues preceding proline is altered considerably; on the other hand, another conformation in which these three residues retain the near-α-helical A-conformational state (with up to 26° changes of their dihedral angles ? and ψ, and a 48° change in one ω from those of the ideal α-helix) has a considerably higher energy. These conclusions are not altered by the substitution of other residues in the place of the Ala preceding Pro. The conformations of the peptide chain next to prolyl residues in or near an α-helix have been analyzed in 58 proteins of known structure, based on published atomic coordinates. Of 331 α-helices, 61 have a Pro at or next to their N-terminus, 21 have a Pro next to their C-terminus, and 30 contain a Pro inside the helix. Of the latter, 16 correspond to a break in the helix, 9 are located inside distorted first turns of the helix, and 5 are parts of irregular helices. Thus, the reported occurrence of prolyl residues next to or inside observed α-helices in proteins is consistent with the computed steric and energetic requirements of prolyl peptides.     

Analysis of ent-kaurenoic acid by ultra-performance liquid chromatography-tandem mass spectrometry

ent-Kaurenoic acid (KA) is a key intermediate connected to a phytohormone gibberellin. To date, the general procedure for quantifying KA is by using traditional gas chromatography–mass spectrometry (GC–MS). In contrast, gibberellins, which are more hydrophilic than KA, can be easily quantified by liquid chromatography-tandem mass spectrometry (LC–MS/MS). In this study, we have established a new method to quantify KA by LC–MS/MS by taking advantage of a key feature of KA, namely the lack of fragmentation that occurs in MS/MS when electrospray ionization (ESI) is in the negative mode. Q1 and Q3 were adopted as identical channels for the multiple reaction monitoring of KA. The method was validated by comparing with the results obtained by selected ion monitoring in GC–MS. This new method could be applicable for the quantification of other hydrophobic compounds.     

Synthesis and conformation of a polyoxyethylene-bound undecapeptide of the alamethicin helix and (2-methylalanyl-L-alanine)1–7

The stepwise synthesis and conformational studies of the N-terminal helical partial sequence of the membrane-modifying polypeptide antibiotic alamethicin are described. The polyoxyethylen esters of the fragments N-t-Boc-L -Pro-Aib-Ala-Gln-Aib-Val-Aib-Gly-OH and N-Ac-Aib-L -Pro-Aib-Ala-Aib-Ala-Gln-Aib-Val-Aib-Gly-OH are synthesized using polyoxyethylene (molecular mass 10,000) as solubilizing support. CD spectra of each intermediate in ethanol show α-helix formation of the N-protected peptide polymers beginning with the nonapeptide and of the N-protonated sequences beginning with the decapeptide. Compared to the helix of alamethicin, temperature- and solvent-dependent CD measurements indicate analogous conformational behavior. The results suggest that in lipophilic media the alamethicin helix can extend the full length of the partial sequence between the two proline residues and that aqueous media favor an increase of random-coil conformation. For model studies of the particular lipid interaction of alamethicin, the stepwise synthesis of peptides with the alternating (Aib-L -Ala)_n sequence (n = 1–7) was carried out on a polyoxyethylene support (molecular mass 6000). CD and ORD studies in ethanol showed a change from the random coil to a right-handed α-helix with increasing peptide length. This change is observed for the N-protected peptides at a chain length of 8 residues and for the N-protonated peptides at a length of 9 residues. The comparison of the CD data of free and polyoxyethylene-bound peptides revealed that the solubilizing polymeric support cannot induce conformational changes. The intensities of the CD bands of t-Boc-(Aib-L -Ala)_n-OPOE (n ≥ 6) are higher than those of alamethicin, and these model peptides show similar temperature and solvent inducible changes of their helix contents.     

Biophysical studies on fragments of the α-factor receptor protein

The receptor for the α-factor mating pheromone of the yeast Saccharomyces cerevisiae consists of 431 amino acid residues and is a member of a family of membrane proteins predicted to have seven transmembrane helices. Fragments of the receptor corresponding to two of the transmembrane helices [residues 246–269 (M6) and 273–302 (M7)], two of the interhelical loops [residues 107–125 (E2) and 191–206 (E3)], and to a portion of the carboxyl terminus [residues 350–372 (CT)] were synthesized using solid-phase methodologies and purified to near homogeneity. CD was used to characterize the secondary structure of these peptides in trifluoroethanol (TFE), in TFE/water mixtures, in sodium dodecyl sulfate (SDS), and in the presence of dimyristoyl phosphatidylcholine (DMPC) liposomes. In TFE, M6 and M7 exhibited CD spectra consistent with highly helical peptides, whereas CT was partially helical. In contrast, E2 and E3 were either disordered or aggregated in this solvent. M6 did not partition well into DMPC vesicles whereas M7 remained helical. Both M6 and M7 assumed helical conformations in 25 mM SDS. The loop neptides and the carboxyl terminus peptide were either in a β-structure or disordered in the presence of lipid. These findings represent the first biophysical evidence for conformations assumed by specific segments of the STE2 receptor protein. © 1994 John Wiley & Sons, Inc.     

Conformational properties of the proline region of porcine neuropeptide Y by CD and 1H-NMR spectroscopy

We synthesized porcine neuropeptide Y (pNPY) N-terminal fragments by solid-phase synthesis techniques and analyzed them for solution Conformational properties by CD and ¹H-nmr spectroscopy. The analogues pNPY_1–9 and pNPY_1–14 displayed CD spectra indicative of random structures and showed no evidence for induced α-helical structures in trifluoroethanol (TFE) up to 50%. However, the CD spectra of pNPY_1-9 suggested a Conformational shift in tetrahydrofuran. Although in aqueous solution the CD spectra of pNPY_1–21 indicated random structures with induction of only a small percentage of α-helix in aqueous TFE, pNPY_1-25 displayed 13% a-helical structure in aqueous solution that increased to 40 and 41% by the addition of TFE and methanol, respectively. The nmr spectra of pNPY_1-9 and the proline region of pNPY_1–25 indicated extended structures with all-trans conformers at Pro5 and Pro8 for pNPY_1–9 and at Pro5, Pro8, and Pro13 for pNPY_1–25; in each case the Tyrl-Pro2 amide bond was in both cis and trans conformations. However, observed nuclear Overhauser effect correlations and UN exchange experiments indicated an α-helical segment in pNPY_1–25 initiated by Pro 13 and extending from residues 14 to 25. Thus, the N-terminal polyproline region of NPY has no propensity to fold into a regular secondary structure, although Pro 13 is a helix initiator, a result consistent with the proposed role of this amino acid in the NPY structural model. © 1995 John Wiley & Sons, Inc.     

Design and synthesis of novel nonpolar host peptides for the determination of the 310- and α-helix compatibilities of α-amino acid buildig blocks: An assessment of α,α-disubstituted glycines

The present work describes three novel nonpolar host peptide sequences that provide a ready assessment of the 3₁₀- and α-helix compatibilities of natural and unnatural amino acids at different positions of small- to medium-size peptides. The unpolar peptides containing Ala, Aib, and a C-terminal p-iodoanilide group were designed in such a way that the peptides could be rapidly assembled in a modular fashion, were highly soluble in solvent mixtures of triflouroethanol and H₂O for CD- and two-dimensional (2D) nmr spectroscopic analyses, and showed excellent crystallinity suited for x-ray structure analysis. To validate our approach we synthesized 9-mer peptides 79a–96 (Table IV), 12-mer peptides 99–110c (Table V), and 10-mer peptides 120a–125d and 129–133 (Table VI and Scheme 8) incorporating a series of optically pure cyclic and open-chain (R)- and (S)-α,α-disubstituted glycines 1–10 (Figure 2). These amino acids are known to significantly modulate the conformations of small peptides. Based on x-ray structures of 9-mers 79a, 80, and 87 (Figures 4–7), 10-mers 124c, 131, and 132 (Figures 9–12), and 12-mer peptide 102b (Figure 13), CD spectra of all peptides recorded in acidic, neutral, and basic media and detailed 2D-nmr analyses of 9-mer peptide 86 and 12-mer 102b, several interesting conformational observations were made. Especially interesting results were obtained using the convex constraint CD analysis proposed by Fasman on 9-mer peptides 79a–d, 80, 81, 86, and 87, which allowed us to determine the relative content of 3₁₀- and α-helical conformations. These results were fully supported by the corresponding x-ray and 2D-nmr analyses. As a striking example we found that the (S)- and (R)-β-tetralin derived amino acids (R)- and (S)-1 show excellent α-helix stabilisation, more pronounced than Aib and Ala. These novel reference peptide sequences should help establish a scale for natural and unnatural amino acids concerning their intrinsic 3₁₀- and α-helix compatibilities at different positions of medium-sized peptides and thus improve our understanding in the folding processes of peptides. © 1997 John Wiley & Sons, Inc. Biopoly 42: 575–626, 1997     

Fast atom bombardment mass spectrometric characterization of peptides

Structural characterization of peptides in the range of 500–5000 Da, using fast atom bombardment (FAB) and Cs⁺ ion liquid secondary ion mass spectrometry (SIMS), is reviewed. These include syntheitc peptides Kemptamide (mol wt 1516); GIF-C15 (mol wt 1875), an isolated natural product as an acylated pentapeptide; and polypeptides generated from enzymatic digests of proteins. MS data is shown to reveal molecular weight and sequence information as well as determine disulfide bonds between cysteine residues and glycosylation sites in the case of a glycopeptide. The complementarity of MS technique to classical biochemical methods for peptide characterization is highlighted. The reader is briefly acquainted with two newer ionization techniques namely, electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI). Synthetic chemists and biochemists can refer to the in-depth review articles that are cited throughout this article.     

A combined biochemical, biophysical and immunological approach towards the identification of celiac disease-specific wheat antigens

Celiac disease (CD) is an inflammatory affliction of the small bowel caused by an immunological hypersensitivity to ingested wheat antigens affecting almost 1 % of the population. The gliadin fraction of wheat has been shown to contain the pathogenic antigens which react with antibodies and T cells. However, there is only limited knowledge regarding the precise nature of the wheat antigens recognized by IgA antibodies from CD patients and diagnostic tests based on the gliadin fraction have been demonstrated to give frequently false positive results. The aim of this study was the characterization of wheat antigens specifically recognized by IgA antibodies of CD patients. We developed a combined biochemical, biophysical, and immunological approach for the identification of celiac disease-specific wheat antigens. It is based on sub-fractionation of the wheat gliadin fraction using two ion exchange chromatography steps, the localization of CD-specific antigens by immunoblotting with IgA antibodies from CD patients, subsequent digestion followed by electro spray ionization–liquid chromatography/mass spectrometry (LC–ESI–MS/MS) and N-terminal sequencing by Edman degradation. Through the sub-fractionation procedure it was possible to separate CD-specific IgA-reactive wheat antigens from other wheat antigens which were also recognized by IgA antibodies of individuals without CD or by CD patients on gluten-free diet. Analysis by LC–ESI–MS/MS and N-terminal sequencing of the sub-fractions and the proteins specifically recognized by CD patients identified certain γ-gliadins with molecular mass of 37,000 and 45,000 as CD-specific wheat antigens. The CD-specific γ-gliadins with the molecular mass of 37,000 and 45,000 should be useful to study pathomechanisms of the disease and to improve the specificity of diagnostic tests for CD.