Increased interest, awareness and research surrounding gluten-related illnesses (such as CD) over the last two decades have resulted in a wealth of new information. New data ultimately provides health care professionals the means to be able to determine proper diagnoses for patients that present with and without gluten- related symptoms. With respect to CD, there is a growing understanding of the pathogenesis of the disease, new methods for screening patients and novel
methodologies for studying gluten proteins and screening gluten-containing foods and products.
Nonetheless, the analysis, characterization and quantification of cereal grain gluten peptides and proteins are complicated. Protein composition, profile and natural sequence fluctuations of grains are in a constant state of flux, due to variances in cultivars, areas grown, climate and the emergence of genetic
engineering. Gluten protein composition can also change during various processing stages of manufacturing. Several well-established analytical techniques, such as SDS-PAGE, capillary electrophoreses, PCR, RP-HPLC and mass spectrometry, have all been used quite successfully, to study cereal grain proteins [46 - 48]. Mass spectrometry has recently become involved in both genomic and proteomic areas of gluten analysis [49]. MS-based methods have focused mainly on applications
involving the qualitative detection and characterization of gluten proteins, through the use of MALDI-TOF-MS [50, 51] and HPLC-MS based methods [26, 52]. Research in the area of assessing the total gluten content and detecting gluten contamination in food has been traditionally done using immunochemical methods [15, 45, 53 - 55]. In fact, the only current commercially available methods for determining the presence of gluten in foods are the immunological antibody-based ELISA methods. Thus, immunochemistry is the only analytical technique that is currently being endorsed by both the FDA and the Codex Alimentarius, for gluten detection in commercially available foods and consumer products.
1.5.1 Measurement of gluten using HPLC-MS
To date, the analytical capabilities of HPLC-MS have found limited application in the area of quantitative detection of trace levels of physiologically relevant gluten peptides in complex matrices. My research was directed toward this end, by the proposed identification of some immunostimulatory gluten peptides, then evaluating commercially available food and consumer products for their presence by
quantitative detection using HPLC-MS. It should be emphasized that this work did not focus on determining the total gluten content in samples, nor the fingerprinting of samples for a gliadin / glutenin profile, nor the identification of which gluten proteins were present in samples. My work did endeavor to develop a means to
comprehensively, selectively and accurately screen complex samples in order to provide evidence of the presence of trace levels of immunogenic gluten.
High performance liquid chromatography (HPLC), atmospheric pressure electrospray ionization (ESI)-ion trap mass spectrometry (ITMS) and triple
quadrupole mass spectrometry (QQQ-MS) are powerful analytical techniques. Their application to the area of proteomics in order to enable peptide and protein
identification, as well as precise quantitative analysis, has been well documented [56 - 59].
Hundreds of gluten peptides are generated by the in-vivo gastric/pancreatic enzymatic digestion of wheat gluten. In order to study these peptides, a similar digestion process could be performed in an in-vitro fashion. An HPLC separation of the resulting mixture of peptides would offer a first degree of specificity needed to study the components of such a complex mixture. ESI-MS detection offers a second
degree of specificity, by providing the ability to detect some specific mass-to-charge (m/z) ions that represent the molecular weights of these gluten peptides. Multiple stages of mass spectrometry (MSn, where n=number of stages) offer even more degrees of specificity, based on the ability to detect product ions generated from the collision-induced dissociation (CID) of certain chosen parent ions. This provides the means to gather the essential information needed to determine the partial or even the complete primary amino acid sequence, thus the identity, of some gluten peptides.
There are several configurations of commercially available instruments that can offer multiple stages of mass spectrometry and are based on combinations of quadrupoles, ion traps, time of flight and magnetic/electric sector mass analyzers. Two of the most common instrument configurations for conducting MSn experiments are the ion trap and triple quadruplole mass spectrometers. These two instrument configurations in combination with HPLC were used in this research and are briefly described as follows.
1.5.1.1 Ion trap mass spectrometer
A schematic of the Agilent Technologies ion trap mass spectrometer is shown in Figure 1.7. The HPLC effluent is nebulized and charged in the atmospheric
pressure electrospray chamber, thus producing charged droplets. These droplets eject ions which are sampled through the capillary and then directed through a series of skimmers, octopole and lenses directly into the trap, while the neutral gas molecules are pumped away. The ability to generate MS and MSn spectra using the
ion trap is based upon computer control of voltages on the ring electrode and voltages and frequencies on the two end caps of the trap.
An ideal application of ion trap mass spectrometry is the structural elucidation of unknown compounds, such as immunogenic gluten peptides. The advantage of using this instrument in this type of application stems from the ability of the trap to accumulate all the ions from the sample being analyzed, simultaneously. This enhances the duty cycle of the ions being studied, as compared to the duty cycle of a scanning mass analyzer, such as a quadrupole. This translates into good full scan sensitivity, which is necessary for identifying and studying unknowns. Another useful feature of the ion trap system is its ability to perform a data dependent acquisition. Thus, MSn spectra can be acquired in addition to full scan spectra, therefore providing much more information which is useful for the identification of unknown compounds.
1.5.1.2 Triple quadrupole mass spectrometer
A schematic of the Agilent Technologies triple quadrupole mass spectrometer is shown in Figure 1.8. The process of generating ions in the electrospray ionization chamber of this instrument is identical to that of the ion trap system, however, the triple quadrupole system transmits the ions directly into a first stage quadrupole mass analyzer rather than a trap analyzer. In this first quadrupole, desired target ions are selected and transported into the next stage of the system, a collision cell. This hexapole collision cell breaks apart the target ions into characteristic product ions and transports them into the final quadrupole analyzer. Here, the product ions are isolated and then directed into the detector assembly.
Triple quadrupole mass spectrometry is ideally suited for the application of quantitative detection of target compounds. Therefore, once the identity of an immunogenic gluten peptide has been determined, this analytical system is well suited to determine its presence and concentration in a food sample.