Mass spectrometry analysis of gliadins in celiac disease

In recent years, scientific research on wheat gluten proteins has followed three main directions aimed at (1) finding relationships between individual genetic alleles coding for gliadins, high or low molecular weight glutenin subunits, and the viscoelastic dough properties of flour-derived products such as pasta and bread; (2) identifying prolamins and derived peptides involved in celiac disease, a pathological condition in which the small intestine of genetically predisposed individuals is reversibly damaged; and (3) developing and validating sensitive and specific methods for detecting trace amounts of gluten proteins in gluten-free foods for celiac disease patients. In this review, the main aspects of current and perspective applications of mass spectrometry and proteomic technologies to the structural characterization of gliadins are presented, with focus on issues related to detection, identification, and quantification of intact gliadins, as well as gliadin-derived peptides relevant to the biochemical, immunological, and toxicological aspects of celiac disease.     

Ambient ionisation mass spectrometry for in situ analysis of intact proteins

Ambient surface mass spectrometry is an emerging field which shows great promise for the analysis of biomolecules directly from their biological substrate. In this article, we describe ambient ionisation mass spectrometry techniques for the in situ analysis of intact proteins. As a broad approach, the analysis of intact proteins offers unique advantages for the determination of primary sequence variations and posttranslational modifications, as well as interrogation of tertiary and quaternary structure and protein‐protein/ligand interactions. In situ analysis of intact proteins offers the potential to couple these advantages with information relating to their biological environment, for example, their spatial distributions within healthy and diseased tissues. Here, we describe the techniques most commonly applied to in situ protein analysis (liquid extraction surface analysis, continuous flow liquid microjunction surface sampling, nano desorption electrospray ionisation, and desorption electrospray ionisation), their advantages, and limitations and describe their applications to date. We also discuss the incorporation of ion mobility spectrometry techniques (high field asymmetric waveform ion mobility spectrometry and travelling wave ion mobility spectrometry) into ambient workflows. Finally, future directions for the field are discussed.     

Identification of proteins from Escherichia coli using two-dimensional semi-preparative electrophoresis and mass spectrometry

Escherichia coli is a gram-negative bacterium that causes sepsis and infections of the nervous system, and the digestive and urinary tracts. The availability of the complete nucleotide sequence encoding the E. coli K-12 genome has made this organism an excellent model for proteomic studies. Semi-preparative two-dimensional electrophoresis, including liquid phase isoelectric focusing (IEF), one-dimensional sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and gel elution, have for the first time been used in combination with matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS), electrospray tandem mass spectrometry and database searching for rapid separation of proteins from a uropathogenic strain of E. coli. The identity of 30 proteins, including the membrane protein nmpC, was obtained using this approach.     

Determination of wheat quality by mass spectrometry and multivariate data analysis

Multivariate analysis has been applied as support to proteome analysis in order to implement an easier and faster way of data handling based on separation by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. The characterisation phase in proteome analysis by means of simple visual inspection is a demanding process and also insecure because subjectivity is the controlling element. Multivariate analysis offers, to a considerable extent, objectivity and must therefore be regarded as a neutral way to evaluate results obtained by proteome analysis.Proteome analysis of storage proteins from the wheat gluten complex based on two-dimensional electrophoresis and analysis of the N-terminal sequence has revealed a protein homologous to gamma-gliadins, tentatively associated with quality and within the molecular weight range 27-35 kDa. Further examinations of gliadin data based on mass spectrometry revealed that quality among wheat varieties could be determined by means of principal component analysis. Further examinations by interval partial least squares made it possible to encircle an overall optimal molecular weight interval from 31.5 to 33.7 kDa. The use of multivariate analysis on data from mass spectrometry has thus shown to be a promising technique to minimize the number of two-dimensional gels within the field of proteome analysis.     

Analysis of proteins in the spent culture medium of Lupinus albus by electrospray ionisation tandem mass spectrometry

Lupinus albus cell cultures secrete a large set of hydrolases into their medium with a small number of highly abundant proteins. We have investigated the protein composition of the medium with two different methods, two-dimensional gel electrophoresis-electrospray ionisation tandem mass spectrometry (ESI-MS-MS) and enzymatic analysis. The proteomic approach revealed the presence of several abundant proteins that had been overlooked using standard enzyme assays, e.g. subtilisin-like protease, glucan 1,3-beta-glucosidase, alpha-amylase, chitinase, thaumatin-like protein, and a secretory pathogenesis-related protein. Several low-abundant proteins were readily detectable by enzymatic assays (peroxidases, phosphatase), but could not be found by ESI-MS-MS. Both data sets support the assumed lytic function of the medium, which appears to be similar to that of the plant vacuole.     

Preparation and characterization of enzymatically hydrolyzed prolamins from wheat,rye, and barley as references for the immunochemical quantitation of partially hydrolyzed gluten

Celiac disease (CD) is a permanent gastrointestinal disorder characterized by the intolerance to a group of proteins called gluten present in wheat, rye, barley, and possibly oats. The only therapy is a strict lifelong gluten-free diet. The standard method for gluten determination in foods produced for CD patients is the R5-enzyme-linked immunosorbent assay (ELISA) as proposed by the recent Codex Alimentarius Draft Revised Standard. This test is based on the determination of prolamins, the alcohol-soluble proteins of gluten, and is available as a sandwich ELISA for intact proteins and as a competitive ELISA for gluten-derived peptides. While the suitability of the sandwich ELISA including a wheat prolamin (gliadin) reference for calibration has been shown by various studies and a ring test, the competitive ELISA still lacks a convenient reference for the quantitation of gluten peptides in fermented cereal foods (e.g., sourdough products, starch syrup, malt extracts, beer). Therefore, the aim of the present study was to prepare a suitable reference for the quantitation of partially hydrolyzed gluten in fermented wheat, rye, and barley products. The prolamin fractions from barley (hordein) and rye (secalin) were isolated from corresponding flours by means of a modified preparative Osborne fractionation. The prolamin fraction from wheat was obtained as reference gliadin from the Prolamin Working Group. The prolamin fractions were successively digested by pepsin and trypsin or pepsin and chymotrypsin procedures, which have been used for CD-specific toxicity tests on cereal storage proteins for many years. The protein/peptide content (N × 5.7) of the prolamin fractions and digests, which was the basis for the calculation of the gluten content by means of ELISA, varied between 67.1% and 96.0%. The prolamin fractions and enzymatic digests were then tested for their response in both sandwich and competitive assays. Intact prolamins responded similarly in both ELISA showing no important differences between the cereals. In the case of digested proteins, however, the sandwich ELISA was considerably less sensitive than the competitive ELISA. The former provided approximately 40% and the latter 70% of the signal intensity obtained with the intact prolamins. Thus, the combination of the competitive ELISA and the enzymatic digests of prolamin fractions as reference was considered to be an adequate system for the analysis of partially hydrolyzed gluten. The limit of detection using a peptic-tryptic hordein digest as reference was 2.3 μg prolamin equivalent per milliliter, and the limit of quantitation was 6.7 μg prolamin equivalent per milliliter. This system was applied for the determination of gluten equivalents in five commercial beverages based on fermented cereals.      

Improved procedure for protein binder analysis in mural painting by LC-ESI/Q-q-TOF mass spectrometry: detection of different milk species by casein proteotypic peptides

Diagnostic techniques applied to the field of cultural heritage represent a very important aspect of scientific investigation. Recently, proteomic approaches based on mass spectrometry coupled with traditional spectroscopic methods have been used for painting analysis, generating promising results for binder’s protein identification. In the present work, an improved procedure based on LC-ESI/Q-q-TOF tandem mass spectrometry for the identification of protein binders has been developed for the molecular characterization of samples from an early-twentieth-century mural painting from the St. Dimitar Cathedral in Vidin, Bulgaria. The proteomic investigation has led to the identification of both egg white and egg yolk proteins, according to traditional old recipes for tempera paintings. In addition, beyond the egg components, the presence of caseins was also revealed, thus suggesting the use of milk as binding medium, fixative or stabilising agent. Furthermore, for the first time, the capability to discriminate the milk origin on the basis of alpha casein proteotypic peptides is reported, that are diagnostic for a given species, thus opening interesting perspectives in art and archaeological fields.     

Analysis of peptides and protein digests by reversed phase high performance liquid chromatography–electrospray ionisation mass spectrometry using neutral pH elution conditions

In this study, the advantages of carrying out the analysis of peptides and tryptic digests of proteins under gradient elution conditions at pH 6.5 by reversed-phase liquid chromatography (RP-HPLC) and in-line electrospray ionisation mass spectrometry (ESI-MS) are documented. For these RP separations, a double endcapped, bidentate anchored n-octadecyl wide pore silica adsorbent was employed in a capillary column format. Compared to the corresponding analysis of the same peptides and protein tryptic digests using low pH elution conditions for their RP-HPLC separation, this alternative approach provides improved selectivity and more efficient separation of these analytes, thus allowing a more sensitive identification of proteins at different abundance levels, i.e. more tryptic peptides from the same protein could be confidently identified, enabling higher sequence coverage of the protein to be obtained. This approach was further evaluated with very complex tryptic digests derived from a human plasma protein sample using an online two-dimensional (2D) strong cation-exchange (SCX)-RP-HPLC-ESI-MS/MS system. Again, at pH 6.5, with mobile phases of different compositions, improved chromatographic selectivities were obtained, concomitant with more sensitive on-line electrospray ionisation tandem mass spectrometric (ESI-MS/MS) analysis. As a consequence, more plasma proteins could be confidently identified, highlighting the potential of these RP-HPLC methods with elution at pH 6.5 to extend further the scope of proteomic investigations.     

Proteomics as an Emergent Tool for Identification of Stress-Induced Proteins in Control and Genetically Modified Wheat Lines

Proteomic methods have been used to identify stress-induced proteins that may have a special role in food science. A new challenge for proteomics has recently been recognised pointing out differences in food protein analysis relevant for nutrition. In this study application of proteomics for traceability of the effect of environmental changes on wheat proteins are addressed. The proteomic analysis involves excision of proteins of interest from two-dimensional (2D) gels, followed by reduction and digestion using trypsin in situ in the spot. The peptides are then analysed using MALDI (matrix assisted laser desorption/ionisation) mass spectrometry and identified by protein databases. The protein set of drought stressed wide-rage herbicide resistant transgenic spring wheat lines: 'T-117', 'T-106-3/a' and 'T-128' and of drought stressed non-transgenic (parent) spring wheat line: 'CY-45' was studied by 2-DE. The drought affected protein expression mostly for the low molecular weight, putative stress-induced proteins were observed in the molecular weight range 15–27 kDa at pH 6,5–7,5. The differentially expressed proteins of albumin and globulin fractions were digested from the gel and digested by trypsin. Number of inhibitor-like proteins were most dominant in the stressed transgenic lines: alpha-amylase/ trypsin inhibitor CM1 precursor, alpha-amylase inhibitor, endogenous alpha-amylase/subtilisin inhibitor (WASI) and a 27 K protein, suggesting that the examined transgenic lines were the sensitive to drought stress.

     

Qualitative and quantitative analysis of wheat gluten proteins by liquid chromatography and electrospray mass spectrometry

Based on analysis by liquid chromatography/electrospray ionisation mass spectrometry, we have developed a new method for fast and sensitive fingerprinting of gliadins and glutenins in wheat flour. Using this procedure the two protein fractions from seven durum wheat varieties have been analysed by high resolution high performance liquid chromatographic separation coupled to accurate determination of molecular mass. In this way, the molecular mass of the single components from both gliadin and glutenin fractions were measured and more than forty components were detected for each fraction indicating a high heterogeneity. Although the chromatographic profiles were similar, the molecular masses of protein components with similar retention times among the varieties were often different. The difference ranged from a few mass units corresponding to single amino acid substitution(s) up to thousands implying peptide deletion or insertion along the protein chain. Two components representing about a half of the gliadin fraction, e.g. gamma(2)- and gamma(3)-gliadin, were identified through the N-terminal sequence and molecular mass determination. We suggest the use of the high level and the molecular mass of these gliadin components as markers to detect traces of wheat in gluten-free food preparations for celiac patients.     

Archived polyacrylamide gels as a resource for proteome characterization by mass spectrometry

Mass spectrometry was applied to identify protein spots excised from an archived two-dimensional polyacrylamide gel that had been dried and stored for eight years at room temperature. All proteins were successfully identified. Detailed characterization of protein digests by matrix-assisted laser desorption/ionization (MALDI) peptide mapping, nanoelectrospray tandem mass spectrometry and MALDI-quadrupole time-of-flight mass spectrometry revealed no evidence of protein degradation or modifications that could hamper identification of proteins in a sequence database. The experiment with a model protein demonstrated that the pattern of tryptic peptides and the yield of individual peptides were not noticeably changed in the in-gel digest of the archived protein spot compared to the digest of the spot excised from a fresh gel. Thus, the characterization of "archived proteomes" has the potential to advance proteomic research without repeating "wet" biochemistry experiments, that had been perfected in the laboratory years ago.     

Nickel species analysis of human colonic tissue using liquid chromatography,gel electrophoresis and mass spectrometry

Studies to specify metal-binding species, such as metalloproteins that are present in trace amounts in colonic cell cytosol, using chromatographic separation methods in combination with inductively coupled plasma mass spectrometry (ICP-MS) as element-specific detection require an optimised sample preparation regarding the solubilisation of the proteins. Focus should be taken to avoid metal contamination, enzymatic digestion by different proteases and oxidation. In this article different sample preparation methods are studied to find a suitable method for the isolation and characterisation of Ni species previously found in cytosols from normal and malignant tissues of the human colon. The total Ni concentrations of the cytosols were determined as well as the total protein content. Thus, a Ni-containing protein could be isolated from cytosols of malignant human colonic tissues using size-exclusion chromatography with ICP-MS for element-specific detection. Ni-containing species in the molecular mass range from 10,000 to 20,000 Da were found and pre-concentrated. The determination of the molecular mass of the species was performed through online coupling of reversed-phase chromatography with electrospray ionisation quadrupole time-of-flight MS. Using identical chromatographic conditions and ICP-MS the detected protein was shown to contain Ni.     

Separation and identification of photosynthetic antenna membrane proteins by high- performance liquid chromatography electrospray ionization mass spectrometry

Functional proteomics of membrane proteins is an important tool for the understanding of protein networks in biological membranes. Nevertheless, structural studies on this part of the proteome are limited. The present review attempts to cover the vast array of methods that have appeared in the last few years for separation and identification of photosynthetic proteins of thylakoid membranes present in chloroplasts, a good model for setting up analytical methods suitable for membrane proteins. The two major methods for the separation of thylakoid membrane proteins are gel electrophoresis and liquid chromatography. Isoelectric focusing in a first dimension followed by denaturing sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) in a second dimension is an effective way to resolve large numbers of soluble and peripheral membrane proteins. However, it is not applicable for isolation of native protein complexes or for the separation of highly hydrophobic membrane proteins. High-performance liquid chromatography (HPLC), on the other hand, is highly suitable for any type of membrane protein separation due to its compatibility with detergents that are necessary to keep the hydrophobic proteins in solution. With regard to the identification of the separated proteins, several methods are available, including immunological and mass spectrometric methods. Besides immunological identification, peptide mass fingerprinting, peptide fragment fingerprinting or intact molecular mass determination by electrospray ionization mass spectrometry (ESI-MS) or matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) have been shown to be very sensitive and effective. In particular, identification of proteins by their intact molecular mass is advantageous for the investigation of numerous biological problems, because it is rapid and reflects the full sequence of the protein and all its posttranslational modifications. However, intact molecular mass determinations of gel-separated membrane proteins are hampered due to the difficulties in extracting the hydrophobic proteins from the gel, whereas HPLC on-line interfaced with ESI-MS enables the rapid and accurate determination of intact molecular masses and consequently an unequivocal protein identification. This strategy can be viewed as a multidimensional separation technique distinguishing between hydrophobicity in the first dimension and between different mass-to-charge ratios in the second dimension, allowing the separation and identification even of isomeric forms.     

Mass spectrometric behaviour of carboxylated polyethylene glycols and carboxylated octylphenol ethoxylates

Mass spectrometric behaviour of mono- and di-carboxylated polyethylene glycols (PEGCs and CPEGCs) and carboxylated octylphenol ethoxylates (OPECs) are discussed. The tendency for ionisation (deprotonation, protonation and cationisation by alkali metal cations) of carboxylated PEGs was compared with that of non-carboxylated correspondents by using both secondary ion mass spectrometry (SIMS) and electrospray ionisation (ESI). The fragmentation of the PEGCs and CPEGCs is discussed and also compared with their neutral correspondents, PEGs. The B/E mass spectra were recorded, using secondary ion mass spectrometry as a method for generation, for deprotonated and protonated molecules and molecules cationised by alkali metal cations. The fragmentation behaviour of PEGs is found to be different from that of CPEGCs, The presence of carboxylic groups may be confirmed not only by the determination of molecular weights of the ethoxylates studied, but also on the basis of the fragment ions formed. The metastable decomposition of the [OPEC-H](-) ions proceed through the cleavage of the bond between the octylphenol moiety and the ethoxylene chain leading to the octylphenoxy anions. It permits determination of the mass of the hydrophobic moiety of the studied carboxylated alkylphenol ethoxylate. ESI mass spectra recorded in the negative ion mode were found to be more suitable for the determination of the average molecular weight of carboxylated ethoxylates than SI mass spectra.     

Two-dimensional gel electrophoresis/matrix-assisted laser desorption/ionisation mass spectrometry of commercial bovine milk

Proteins in commercial bovine milk have been separated by two-dimensional gel electrophoresis and examined by matrix-assisted laser desorption/ionisation mass spectrometry. Gel separation was conducted in two different pH gradients, 3-10 and 6-11; the latter range resulted in a higher spot resolution and favoured the basic proteins. We have limited the time-of-flight mass spectrometry analysis to the linear mode to examine the capability of reliable relative molecular masses of the intact proteins in their characterisation. The present study draws attention to the difficulty of identifying basic proteins with low molecular masses (below 12000 Da) that are commonly encountered in milk samples.     

Structural characterisation of tyrosine-nitrated peptides by ultraviolet and infrared matrix-assisted laser desorption/ionisation Fourier transform ion cyclotron resonance mass spectrometry

Nitration of tyrosine residues in proteins may occur in cells upon oxidative stress and inflammation processes mediated through generation of reactive nitroxyl from peroxynitrite. Tyrosine nitration from oxidative pathways may generate cytotoxic species that cause protein dysfunction and pathogenesis. A number of protein nitrations in vivo have been reported and some specific Tyrosine nitration sites have been recently identified using mass spectrometric methods. High-resolution Fourier transform ion cyclotron resonance mass spectrometry (MALDI) FT-ICR-MS) is shown here to be a highly efficient method in the determination of protein nitrations. Following the identification of nitration of the catalytic site Tyr-430 residue of bovine prostacyclin synthase, we synthesised several model peptides containing both unmodified tyrosine and 3-nitro-tyrosine residues, using solid-phase peptide synthesis (SPPS). The structures of the nitrotyrosine peptides were characterised both by ESI- and by matrix-assisted laser desorption/ionisation (MALDI)-FT-ICR-MS, using a standard ultraviolet (UV) nitrogen nitrogen laser and a 2.97 microm Nd-YAG infrared laser. Using UV-MALDI-MS, 3-nitrotyrosyl-peptides were found to undergo extensive photochemical fragmentation at the nitrophenyl group, which may hamper or prevent the unequivocal identification of Tyr-nitrations in cellular proteins. In contrast, infrared-MALDI-FT-ICR-MS did not produce fragmentation of molecular ions of Tyr-nitrated peptides.     

Clickable report tags for identification of modified peptides by mass spectrometry

The identification and quantification of modified peptides are critical for the functional characterization of post-translational protein modifications (PTMs) to elucidate their biological function. Nowadays, quantitative mass spectrometry coupled with various bioinformatic pipelines has been successfully used for the determination of a wide range of PTMs. However, direct characterization of low abundant protein PTMs in bottom-up proteomic workflow remains challenging. Here, we present the synthesis and evaluation of tandem mass spectrometry tags (TMT) which are introduced via click-chemistry into peptides bearing alkyne handles. The fragmentation properties of the two mass tags were validated and used for screening in a model system and analysis of AMPylated proteins. The presented tags provide a valuable tool for diagnostic peak generation to increase confidence in the identification of modified peptides and potentially for direct peptide-PTM quantification from various experimental conditions.     

On-line size-exclusion chromatography/electrospray ionisation mass spectrometry of aquatic humic and fulvic acids

Isolated aquatic humic and fulvic acids were analysed with on-line size exclusion chromatography/electrospray ionisation mass spectrometry (SEC/ESI-MS). An eluent composition which enabled electrospray ionisation was identified. The SEC separation improved interpretability of mass spectra and may open up new possibilities for molecular weight determination of humic substances. A linear dose-response relationship over a factor of 20 was obtained and the limit of detection was 50ng/uL for humic and fulvic acids. Spectral changes due to different ionisation conditions (pH and cone voltage) were investigated. A natural water sample from a Swedish lake was analysed. Copyright 2000 John Wiley & Sons, Ltd.     

Analysis of artemisinin by a packed-column supercritical fluid chromatography-atmospheric pressure chemical ionisation mass spectrometry technique

A packed-column supercritical fluid chromatography-atmospheric pressure chemical ionisation mass spectrometry method was studied for the determination of artemisinin from Artemisia annua L. extracts. The technique does not require any kind of derivatisation prior to the analysis. All samples were simply dissolved in methanol and injected into the mobile phase. Detection was achieved by using mass spectrometry with atmospheric pressure chemical ionisation. The ionisation technique is relatively soft and provides protonated molecular ion and informative structural fragmentation for the compound. Benzophenone was used as a chromatographic standard for the determination of the analytical reproducibility. The supercritical carbon dioxide mobile phase used in the system was modified by 10% methanol. The average absolute retention time was 3.54 min with a standard deviation of 0.017 min and a relative standard deviation of 0.4% with respect to benzophenone for the procedure. The correlation coefficient was 0.998 and detection limit 370 pg on column.