A Review of Gelatin Source Authentication Methods <s> Mass Spectrometric Method <s> The epidemic of bovine spongiform encephalopathy (BSE) is thought to have resulted from feeding scrapie-infected sheep to cattle. This has led to a ban of feeding animals with "processed animal protein"(PAP). We report a novel approach for the mass spectrometric detection of PAP contamination in animal feedstuffs by detecting gelatine, a derivative of the major animal protein collagen. A method was developed to hydrolyse gelatine standards with hydrochloric acid, followed by detection of the derived hydrolytic peptides at m/z 828, 915, 957 and 1044 by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and liquid chromatography electrospray ionisation mass spectrometry (LC-ESI-MS-MS). The marker peptides can be detected at concentrations of 100 ng ml(-1) gelatine in water with MALDI. The procedure was adapted to obtain a suitable peptide map of gelatine extracted from spiked feed. The ratio of signal area of the gelatine-derived peptide at m/z 1044 to the internal standard at m/z 556 is shown to relate to the total amount of gelatine present in the sample. <s> BIB001 </s> A Review of Gelatin Source Authentication Methods <s> Mass Spectrometric Method <s> Sequence analysis of bovine and porcine collagen type I indicates that some partial sequences are specific for bovine or porcine collagen type I, which can be used as marker peptides for gelatin differentiation. Bovine and porcine gelatins were digested with trypsin. The digest mixtures were analyzed with high performance liquid chromatography/mass spectrometry ( HPLC/MS ). Marker peptides were identified by the method of peptide sequence tag. The results indicated that the bovine and porcine. gelatins could be differentiated according to the sequences of marker peptides. Further analysis indicated that some prolines in the marker peptides were hydroxylated, which should be taken into account to increase the confidence in peptide identification. The relative abundance of marker peptides in gelatin of different molecular weight range was also investigated. The results indicate that identification of the marker peptides in the digested gelatins by HPLC/MS is an alternative method for differentiation of bovine and porcine gelatins. <s> BIB002 </s> A Review of Gelatin Source Authentication Methods <s> Mass Spectrometric Method <s> Gelatin is a mixture of polypeptides obtained by hydrolysis of collagen primarily from bovine and porcine skin and bones. The similarity between different gelatins makes it difficult to trace their species origin. In this work, a new method for differentiation between bovine and porcine gelatin was developed based on detection and identification of marker peptides in digested gelatins. Sequence alignment analysis indicates that bovine and porcine Type I collagen contain differential sequences. The gelatins were digested by trypsin, and the resulting peptides were analyzed by high performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS). The marker peptides specific for bovine and porcine were successfully detected in the digested bovine and porcine gelatin, respectively. Comparative analysis indicated that more marker peptides could be detected in gelatin with a higher mean molecular weight. It was found that proline hydroxylation was a key factor affecting the peptide identification. For peptides such as GPPGSAGSPGK and GPPGSAGAPGK detected in digested bovine and porcine gelatin, respectively, the sequence should be verified manually since the mass shift caused by proline hydroxylation can be confused with the mass difference between Set and Ala residues. The results indicate that detection of marker peptides in the digested gelatin sample using HPLC-MS/MS is an effective method to differentiate between bovine and porcine gelatin. (C) 2009 Elsevier Ltd. All rights reserved. <s> BIB003 </s> A Review of Gelatin Source Authentication Methods <s> Mass Spectrometric Method <s> Species identification of fragmentary bone, such as in rendered meat and bone meal or from archaeological sites, is often difficult in the absence of clear morphological markers. Here we present a robust method of analysing genus-specific collagen peptides by mass spectrometry simply by using solid-phase extraction (a C18 ZipTip®) for peptide purification, rather than liquid chromatography/mass spectrometry (LC/MS). Analysis of the collagen from 32 different mammal species identified a total of 92 peptide markers that could be used for species identification, for example, in processed food and animal feed. A set of ancient (>100 ka@10°C) bone samples was also analysed to show that the proposed method has applications to archaeological bone identification. Copyright © 2009 John Wiley & Sons, Ltd. <s> BIB004 </s> A Review of Gelatin Source Authentication Methods <s> Mass Spectrometric Method <s> In recent years, mass spectrometry has moved more than ever before into the front line of protein-centered research. After being established at the qualitative level, the more challenging question of quanti fi cation of proteins and peptides using mass spectrometry has become a focus for further development. In this chapter, we discuss and review the strategies and problems of the methods currently in use for the quantitative analysis of peptides, proteins, and fi nally proteomes by mass spectrometry. The common themes, the differences, and the potential pitfalls of the main approaches are presented in order to provide a survey of the emerging fi eld of quantitative, mass spectrometry-based proteomics. <s> BIB005 </s> A Review of Gelatin Source Authentication Methods <s> Mass Spectrometric Method <s> We presented a novel nanoUPLC-MS(E) workflow method that has potential to identify origin of gelatin in some dairy products; yoghurt, cheese and ice cream. In this study, the method was performed in two steps. In the first step, gelatin was extracted from these products before the MS-sample preparation. In the second step, tryptic gelatin peptides were separated and analyzed with ultra-performance liquid chromatography and electrospray ionization quadrupole time-of-flight mass spectrometry (nanoUPLC-ESI-q-TOF-MS(E)). The novelty of this setup was that it functioned in a data independent acquisition mode and that alternate low and elevated collision energy was applied to acquire precursor and product ion information. This enabled accurate mass acquisition on the peptide level to identify the gelatin peptides. The marker peptides specific for porcine and bovine could be successfully detected in the gelatin added to the dairy products analyzed, revealing that the detection of marker peptides in the digested gelatin samples using nanoUPLC-ESI-q-TOF-MS(E) could be an effective method to differentiate porcine and bovine gelatin in the dairy products. <s> BIB006 </s> A Review of Gelatin Source Authentication Methods <s> Mass Spectrometric Method <s> Gelatine is a component of a wide range of foods. It is manufactured as a by-product of the meat industry from bone and hide, mainly from bovine and porcine sources. Accurate food labelling enables consumers to make informed decisions about the food they buy. Since labelling currently relies heavily on due diligence involving a paper trail, there could be benefits in developing a reliable test method for the consumer industries in terms of the species origin of gelatine. We present a method to determine the species origin of gelatines by peptide mass spectrometry methods. An evaluative comparison is also made with ELISA and PCR technologies. Commercial gelatines were found to contain undeclared species. Furthermore, undeclared bovine peptides were observed in commercial injection matrices. This analytical method could therefore support the food industry in terms of determining the species authenticity of gelatine in foods. <s> BIB007
Mass spectrometry (MS) has been found to be very useful for qualitative and quantitative analysis of protein and peptides. Although, it was initially developed to study small molecules, several improvements have recently led to the use of MS in proteomics. These developments have led to different types of MS such as time-offlight (ToF), quadrupole, (Paul) ion trap, FTICR, and orbitrap, as well as, different techniques (matrix-assisted laser desorption/ionization (MALDI) and electrospray ionisation (ESI) that allowed the ionisation of biomolecules (peptides, proteins, and nucleotides) for easy detection by mass spectrometry. Proteomic studies involve a mixture of numerous proteins/peptides that are required to be separated using chromatography techniques such as high-performance column chromatography (HPLC) and ultra-performance column chromatography (UPLC). Hybridisation of different mass spectrometers (MS) combined with HPLC/UPLC, MALDI/ESI has made mass spectrometric methods for protein quantification become more powerful BIB005 . Several studies have been conducted to developed MS-based methods for differential analysis of gelatin source. BIB001 investigated the use of MALDI-TOF MS and LC-ESI-MS-MS for detection of HCl gelatin-derived peptides in animal feed. The derived hydrolytic peptides at m/z 828, 915, 957 and 1044 were analysed by MALDI-TOF MS and LC-ESI-MS-MS. Using MALDI, the sensitivity of the method was reportedly improved to 100 ng mL −1 of peptide solution. It was concluded that some species-specific ions could be used as a marker for the presence of bovine gelatin in animal feed. G.-F. BIB002 and G. BIB003 utilised highperformance liquid chromatography/mass spectroscopy (HPLC/MS) and HPLC-MS/MS, respectively, to analyse trypsin digested bovine and porcine gelatins. Specie-specific (maker) peptides were identified and their sequences could be used for differential analysis of bovine and porcine gelatins. The maker peptides contained hydroxylated prolines that should be considered to increase the confidence of peptide identification. This is because of the possibility of mass shift due to proline hydroxylation can be misleading. BIB004 conducted species identification of bone collagen peptides from 32 different mammal species using MALDI-TOF MS. The peptide obtained using solid-phase extraction showed 92 peptide markers that could be used for species identification in processed food and animal feed. BIB006 presented an ultra-performance liquid chromatography and electrospray ionisation quadrupole time-of-flight mass spectrometry (nanoUPLC-ESI-Q-TOF-MS) based data independent acquisition technique to detect maker peptides for differential analysis of gelatin sources (porcine and bovine) in dairy products. Specie-specific peptides maker were successfully detected in gelatin added to dairy products analysed. The result showed that nanoUPLC-ESI-Q-TOF-MS method is an effective and a rapid way to determine the gelatin origin. BIB007 presented a method to determine the species origin of gelatins by peptide-MS methods (MALDI-TOF and LC-ESI-MS-MS). The peptides were obtained by pepsin digestion of nine commercial gelatins. It was found that the commercial gelatins contained undeclared species and that the method was able to determine not only the origin of gelatin but was also able to differentiate between bovine and porcine as well as other wide range of species (horse, goat, sheep, fish and various poultry species). Hence, it was concluded that the method could support food industry in determining the species authenticity of gelatin in foods. However, the drawbacks of mass spectrometric method include (i) difficulties in detection of peptides marker in digested gelatin due to high level of similarities between gelatin sequence in mammals, (ii) proline hydroxylation pose challenge that make peptide identification more difficult and (iii) the threshold for specific peptide identification varies from species to species BIB006 .