AU2008253573A1 - Fragmentation profile based authentification of protein source - Google Patents

Description

WO 2008/141358 PCT/AU2008/000614 FRAGMENTATION PROFILE BASED AUTHENTIFICATION OF PROTEIN SOURCE Field of the Invention The present invention relates to a method for determining the source of a protein sample from any tissue or liquid that contains proteins such as bacteria, fungi, 5 human and animal tissue, plant material. The invention more specifically relates to a method of typing protein samples from foodstuffs such as meat. Background Art The source of many food products, particularly cooked products, is not easily determined and thus consumers are forced to rely on the honesty of traders 10 when purchasing products. The dwindling stocks (and high market value) of some livestock such as certain fish, have led to the use of alternative species as substitutes. Often, these substitutes are difficult to distinguish from the food product advertised. Furthermore, the increasing demands for quality control, quality assurance and 15 the associated need to trace food through the distribution channel from field to table has created a need for accurate methods of food product identification. Accuracy in labelling is becoming of critical importance both to protect industries and the consumer. Protein based technologies for the identification of samples may be carried out 20 on a single protein (e.g. peptide mass fingerprinting; or protein sequencing) or carried out on a complex mixture of proteins (e.g. protein profiling of a mixture of intact proteins; or proteome identification by identifying protein components of a protein mixture by database matching of individual constituent peptide fragments obtained after enzyme digestion). These methods can be carried out using 25 techniques such as chromatography, mass spectrometry and electrophoresis on, for example, I D gels. However, these existing techniques can lack reproducibility, are difficult to perform, may produce results that require subjective interpretation and may be affected by processing of the sample.

WO 2008/141358 PCT/AU2008/000614 -2 There also exists a number of DNA based identification methods, such as PCR and RFLP based tests. However, such tests require sequence-specific primers, thus requiring that the gene of interest to have been cloned and the samples to undergo amplification. Secondly, the gene must be largely conserved for the 5 PCR amplification to be successful but also polymorphic for the RFLP to show a difference. Furthermore, DNA based methods may be affected by any processing of the sample that leads to degradation of the DNA. A further limitation with the current methods is that they do not provide detailed information regarding the source of a sample. Most of the available tests give a 10 simple yes/no answer, identifying that the sample is or is not from a given source, but they do not necessarily identify the source. The present invention addresses a need in the art for improved methods of tissue identification capable of at least ameliorating one or more of the problems attendant with the prior art. 15 Summary of the Invention The present invention provides a method for determining the source of a protein sample comprising the steps of: (i) isolating a subset of proteins from the sample; (ii) fragmenting one or more of the proteins in the protein subset; 20 (iii) characterising the proteins and protein fragments of the protein subset; and (iv) using the results from step (iii) to determine the origin of the sample. The present invention further provides a method for determining the source of a protein sample comprising the steps of: 25 (i) isolating a subset of proteins from the sample; 0I ID TITI IT" LII""T IDI II I"" )L'% Of"IA I I WO 2008/141358 PCT/AU2008/000614 -3 (ii) fragmenting one or more of the proteins in the protein subset; (iii) characterising the proteins and protein fragments of the protein subset using mass spectrometry; and (iv) using the results from step (iii) to determine the origin of the sample. 5 The present invention additionally provides a method for determining the source of a protein sample comprising the steps of: (i) isolating a subset of proteins from the sample; (ii) fragmenting one or more of the proteins in the protein subset; (iii) characterising the proteins and protein fragments of the protein subset 10 using mass spectrometry; (iv) using the results from step (iii) to prepare a peptide signature; and (v) using the peptide signature from step (iv) to determine the origin of the sample. Brief Description of the Drawings 15 Figure 1 is an image of crude extracts from raw and cooked fish tissue (prior to spin filtration step) electrophoresed on a 10-20% acrylamide BioRad Criterion pre-cast polyacrylamide gel in the presence of sodium dodecyl sulfate (SDS) and stained with Coomassie Brilliant Blue G250. Lane 1: Raw Barramundi [sample 1]; Lane 2: Raw Dhufish; Lane 3: Raw Pearl Perch; Lane 4: Raw Nile Perch; 20 Lane 5: Raw Barramundi [sample 2]; Lane 6: Raw Redspot Emperor; Lane 7: Cooked Barramundi [sample 1]; Lane 8: Cooked Dhufish; Lane 9: Cooked Pearl Perch; Lane 10: Cooked Nile Perch; Lane 11: Cooked Barramundi [sample 2]; Lane 12: Cooked Redspot Emperor. Molecular masses in kDa, derived from BioRad polypeptide mass standards, are shown (Mr); 0I ID TITI ITC eLICCT IDI II n&% ftiA I I WO 2008/141358 PCT/AU2008/000614 -4 Figure 2 is an image of <10 kDa protein extracts from cooked fish tissue (after column desalting step) electrophoresed on a 10-20% acrylamide BioRad Criterion pre-cast polyacrylamide gel in the presence of sodium dodecyl sulfate (SDS) and stained with silver nitrate. Lane 1: Cooked Dhufish; Lane 3: Cooked 5 Redspot Emperor; Lane 5: Cooked Nile Perch; Lane 7: Cooked Pearl Perch; Figure 3 is a flowchart outlining one embodiment of the present invention for determining the source of a raw fish (Barramundi) protein sample. Figure 4 is a flowchart outlining one embodiment of the present invention for determining the source of a raw fish protein sample. 10 Figure 5 is a flowchart outlining one embodiment of the present invention for determining the source of a raw shark protein sample. Figure 6 is a flowchart outlining one embodiment of the present invention for determining the source of a wheat protein sample. Figure 7 is a flowchart outlining one embodiment of the present invention for 15 determining the source of a subclover protein sample. Detailed Description of the Invention Determining the source of a protein sample The present invention provides a method for determining the source of a protein sample comprising the steps of: 20 (i) isolating a subset of proteins from the sample; (ii) fragmenting one or more of the proteins in the protein subset; (iii) characterising the proteins and protein fragments of the protein subset; and 0I IDOTITI IT" LII""""T IDI II I"" -)&% fiA I I WO 2008/141358 PCT/AU2008/000614 (iv) using the results from step (iii) to determine the origin of the sample. The present invention is based on the surprising discovery that a subset of proteins from a sample can be used to reliably and simply determine the source of the sample. 5 The method produces more consistent results relative to techniques that involve the generation of protein profiles because it uses a less complex mixture of proteins and thus overcomes many of the problems associated with the prior art. The method is also more sensitive and thus can be used to distinguish between samples from closely related sources. Furthermore, the method of the present 10 invention is more flexible in that it can be more easily applied to different sample types. The method of the present invention may be applied to any sample that contains protein, such as bacteria, fungi, human and animal tissue, plant material. Preferably, the protein sample is from a food source such as a fungi, plant or 15 animal tissue such as meat. Thus, the present invention also provides a method for determining the source of a food sample comprising the steps of: (i) isolating a subset of proteins from the sample; (ii) fragmenting one or more of the proteins in the protein subset; (iii) characterising the proteins and protein fragments of the protein subset; 20 (iv) using the results from step (iii) to determine the origin of the sample. The food sample may be any tissue that contains protein and is used as a food source for animals and/or humans, including fungi, plant tissue or animal tissue. Preferably, the tissue sample is meat from livestock which are used as a food source for animals and/or humans. More preferably, the meat is from fish, cattle, 25 sheep, goats, pigs or avian species. Even more preferably, the meat is from a fish selected from the group comprising: Lethrinus spp including L. lentjan, Lates spp including L. niloticus and L. calcarifer, Glaucosoma spp including G. 0I IDOTITI IT" LII""T IDI II I"" )&% Of"IAII WO 2008/141358 PCT/AU2008/000614 -6 hebraicum, G. scapulare, G. buergeri, G magnificans, Plectropomus spp such as P. leopardus and members of the Merluciidae family such as various Merluccius spp. Other fish which may be sampled using the method of the present invention 5 include fish such as Ocean Jackets, Redfish, Sea Mullet, Snapper, Dories such as Mirror Dory, Ling such as Pink Ling, Jackass Morwong, Eastern School Whiting, Yellowfin Bream, Silver Trevally and Emperor fish such as Redspot Emperor. The method may also be used to identify tissue from tuna, sharks, Pilchards, Coral Trout, Flathead, Mullet, Barramundi, Spanish mackerel, Orange 10 Roughy, Bream, Salmon, Gemfish and large ocean perchs such as the Patagonian Toothfish. The food sample to be determined using the method of the present invention may be a cooked or raw food sample, eg it may be tissue derived from cooked or raw meat or cooked or raw fungi etc. 15 The term "protein" used herein describes polymers of amino acids and all post translationally modified protein molecules, for example amino acid sequences having undergone, but not restricted to, phosphorylation, sulphation, lipidation, methylation, acetylation, carboxylation, oxidation, ADP (adenosine diphosphate) ribosylation, and glycosylation including the formation of glycoproteins or 20 proteoglycans. The proteins are preferably extracted from the sample prior to isolation. Preferably, this extraction is carried out using a surfactant. Preferably, the surfactant is chosen from the group comprising: zwitterionic detergents such as n-Decyl-N,N-Dimethyl-3-Ammonio-1-Propane-Sulfonate (sulphobetaine 10 or SB 25 3-10) or 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Other zwitterionic detergents that may be used include 3 (Dodecyldimethylammonio)propanesulfonate, 3-(Dodecyldimethylammonio) propanesulfonate, 3-(N,N-Dimethylmyristylammonio)propanesulfonate, 3-(N,N Dimethyloctadecylammonio)propanesulfonate, 3-(N,N-Dimethyloctylammonio) 30 propanesulfonate, and 3-(N,N-Dimethylpalmitylammonio)propanesulfonate. Most preferably, the surfactant used to extract the proteins is SB3-1 0. 0I ID TITI IT"" LII""j""T ID I n"" M ftiA I I WO 2008/141358 PCT/AU2008/000614 -7 The amount of surfactant or detergent used to extract the proteins is preferably between 0.01%v/v and 1.0%v/v. More preferably, the amount used is between 0.04%v/v and 0.3%v/v. Most preferably, 0.05%v/v surfactant or detergent is used. 5 The subset may be isolated using any means apparent to one skilled in the art provided it generates a subset that is capable of being used to determine the source of the sample. Preferably, the subset is isolated by relying on at least one of the following characteristics: size, solubility, charge, isoelectric point, affinity. Preferably, the subset is isolated using at least size as the defining 10 characteristic. Even more preferably, the subset is isolated by relying on at least two of the above characteristics, such as size and solubility. Following isolation, preferably the subset comprises at least 80%, 85%, 90% 95% or 99% of each of the subset proteins that were in the sample prior to isolation. However, the method may be carried out even if there is only, for 15 example, 5% of one of the subset proteins isolated from the sample prior to isolation. It is also most preferable that all of the proteins in the subset that were present in the sample prior to isolation be present in the subset after isolation. Thus, preferably 100% of the subset proteins are present in the isolated subset, 20 although the actual amount of each of those proteins may be small. However, the isolated subset of proteins may comprise less than 100% of the total subset of proteins, providing that the same subset of proteins is always obtained by the isolation method. When the subset is isolated on the basis of size, the size may be varied 25 depending on the sample and more particularly the complexity i.e. nature and number of the proteins in a given sample. For example, the subset may be all the proteins in a sample that have a molecular mass less than a certain point such as 5, 10, 15, 20 or 30kDa. Alternatively, the subset may be all the proteins within a molecular mass range such as range of 1-10, 1-5 or 1-2kDa. For 30 example, the subset may be all the proteins with a molecular mass of between 1 5kDa, 1-1OkDa, 5-1OkDa, 10-15kDa or 15-20kDa. Any other size range may be 0I ID TITI ITI" OLII""I""T IDI II I"" ' M Of"IA I I WO 2008/141358 PCT/AU2008/000614 -8 chosen, as deemed appropriate and suitable for a given application. Most preferably, the subset is formed of all the proteins in a sample that have a molecular mass less than I OkDa. When the subset is isolated on the basis of size the actual technique used to 5 produce the subset can be varied. Preferably, the sample is subjected to a separation technique such as ultrafiltration e.g. centrifugal filtration with or without pressure, gas pressure filtration, tangential flow filtration, chromatographic (gel) filtration, and solvent absorption. Alternatively, other techniques resulting in this separation can be used, e.g. electrophoresis, dialysis. 10 The size separation is preferably carried out using mass column filters or spin columns. When the subset of proteins is isolated based on two characteristics, a first subset may be selected on the basis of solubility and then a second subset of the first subset may be selected on the basis of size. Thus, the first subset of 15 proteins may be chosen to comprise proteins which are water soluble. This subset of <10 kDa proteins may then be selected from the water soluble proteins and characterised. Alternatively, when the subset of proteins is isolated based on two characteristics a first subset may be selected on the basis of size and then a second subset of 20 the first subset may be selected on the basis of solubility. For example, the first subset may be chosen to comprise proteins with a size of less than 10kDa. A further subset of the <10 kDa proteins may then be chosen to comprise proteins that are water soluble. A further subset of <10 kDa water soluble proteins may then be characterised. 25 The subset may be fragmented and characterised in various ways apparent to one skilled in the art. Preferably, the subset is fragmented by further treating the subset to generate protein fragments and then analysing the resulting fragments to characterise the proteins by forming a profile of the protein and protein fragments in the subset. 0I ID TITI IT"" OLII""T IDI II " ')L' Of"IA I I WO 2008/141358 PCT/AU2008/000614 -9 Thus, the subset may be treated to cause fragmentation of one or more, and preferably all, of the proteins in the subset. This fragmentation is carried out to increase the heterogeneity of the peptides in the subset for more sensitive analysis and acts to reduce the proteins to fragments of a size that can be 5 applied to a method such as, for example, chromatography or electrophoresis for further analysis. In this regard, the fragmentation of the protein subset also generates a large, but preferably manageable number of informative fragments from the various protein members of the protein subset. A protein from the subset may be fragmented by relying on different strengths of 10 bonds between amino acids in the protein sequence and/or differences in the actual amino acid sequence. Thus, the number and size of the fragments generated from each protein of the protein subset will be characteristic of the source of the protein sample. Fragmentation may be achieved by any method that is precise and produces 15 reproducible protein fragments. Preferably, enzymes are used to digest the proteins. Most preferably, the enzyme used to digest the proteins into fragments is trypsin. Alternatively, the enzyme selected to digest the protein subset into fragments may be one chosen to digest the proteins at regions that have inherent genetic variability, so that the pattern of fragments generated depends 20 on whether a given protein from a given source has sites that may be cleaved by the protein. Enzymes which are commonly used for restriction fragment length polymorphism (RFLP) fragmentation of proteins may be used in the method of the present invention. Alternatively, if conditions are chosen such that the fragmentation of the protein subset is precise and reproducible, methods such as 25 slow highly charged ion impact, or hydroxyl radical activated cleavage may be used to fragment the proteins. The subset may be characterised by one or more techniques selected from the group consisting of: chromatography, for example high-performance liquid chromatography (HPLC), gas chromatography (GC), liquid chromatography (LC), 30 mass spectrometry (MS); or electrophoresis, for example I D or 2D gels; mass spectrometry using matrix assisted laser desorption ionisation (MALDI); mass 0I IDOTITIIT"" OLII""T IDI II " 0 m n f% iA II WO 2008/141358 PCT/AU2008/000614 -10 spectrometry using electrospray or other methods known to the skilled person. Preferably, the subset is characterised using mass spectrometry. Alternatively, more than one characterisation technique may be used to characterise the sample. For example, the subset may be characterised by a combination of LC 5 and MS. Thus, the present invention also provides a method for determining the source of a protein sample comprising the steps of: (i) isolating a subset of proteins from the sample; (ii) fragmenting one or more of the proteins in the protein subset; 10 (iii) characterising the proteins and protein fragments of the protein subset using mass spectrometry; and (iv) using the results from step (ii) to determine the origin of the sample. Once the subset has been characterised, these results can be used to determine the origin of the sample. Preferably, the results are compared to a database 15 containing at least one reference result indicative of an identity. When chromatography is used, it is preferable to select a portion of the spectrometric peaks or gel electrophoresis spots which can then be compared between samples. For example, the 50-100 peaks or spots with the strongest signal (i.e. the highest peak or the brightest spot) may be chosen. These 20 selected fragments can then be used to form a characteristic peptide signature of the sample. When electrospray mass spectrometry is used, it is preferable to select a portion of the spectrometric peaks that are multiply charged (ie with a charge of +2 or higher) which can then be compared between samples. For example, the 50 25 100 multiply charged ions with the strongest signal (i.e. the highest peak) may be chosen. When MALDI mass spectrometry is used, it is preferable to select a portion of the spectrometric peaks with the strongest signal that are not matrix 0I IDTITI ITI"" 0LII""T IDIII I"" )&% f"IA II WO 2008/141358 PCT/AU2008/000614 - 11 peaks (i.e. the highest peptide peaks) which can then be compared between samples. These selected fragments can then be used to form a characteristic peptide signature of the sample, which can be compared between samples. Thus, the present invention also provides a method for determining the source of 5 a protein sample comprising the steps of: (i) isolating a subset of proteins from the sample; (ii) fragmenting one or more of the proteins in the protein subset; (iii) characterising the proteins and protein fragments of the protein subset using mass spectrometry; 10 (iv) using the results from step (iii) to prepare a peptide signature; and (v) using the peptide signature from step (iv) to determine the origin of the sample. Preferably, the signature prepared in step (iv) has less than 50 peaks and even more preferably about 10 peaks. 15 The method of the present invention may further comprise sequencing of the peptides in the signature. This sequencing may be carried out after identification of the peptide signature, to sequence the peptides that make up the peptide signature. Alternatively sequencing may comprise fragmentation of the peptides to produce a mass fragmentation pattern indicative of amino acid sequence. 20 Database It may be desirable to develop a database of peptide signatures that can be used to identify the source of a sample. Thus, the present invention also provides a method for preparing a database of peptide signatures comprising the steps of: (i) isolating a subset of proteins from a protein sample; 0I IDOTITI IT" LII""""T IDI II I"" -)&% fiA I I WO 2008/141358 PCT/AU2008/000614 - 12 (ii) fragmenting one or more of the proteins in the protein subset; (iii) characterising the proteins and protein fragments of the protein subset; and (iv) placing the results from step (iii) into a database. 5 The database can be used as a reference source for new samples analysed using the method of the present invention. Thus, samples can be compared or contrasted with the database with a view to determining the source of a sample or the relatedness of a sample to reference sources. Method of identifying a subset for use in the method of the present invention 10 The method of the present invention relies on the determination of subsets that are sufficiently indicative of sample source. Not all subsets will be suitable for use in the methods described herein. Preferably, the subset is the simplest, in terms of the number of proteins it contains, which still enables reliable determination of the source of a sample. 15 One way of conveniently determining a subset for use in the present invention involves analysing the total proteins in the sample prior to selecting a subset of the proteins. Preferably, the proteins in the sample are graphically displayed following electrophoresis or mass spectrometry and this is used to select a subset of proteins. For example, it may be advantageous to display all the 20 proteins in the sample and then view the displayed proteins or portions thereof to determine which subset of proteins has high variability or polymorphism. General Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically 25 described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in the specification, 0I ID TITI IT" LII""T IDI II I"" ')l n f% iA I I WO 2008/141358 PCT/AU2008/000614 -13 individually or collectively and any and all combinations or any two or more of the steps or features. The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. 5 Functionally equivalent products, compositions and methods are clearly within the scope of the invention as described herein. The entire disclosures of all publications (including patents, patent applications, journal articles, laboratory manuals, books, or other documents) cited herein are hereby incorporated by reference. No admission is made that any of the 10 references constitute prior art or are part of the common general knowledge of those working in the field to which this invention relates. As used herein the term "derived" and "derived from" shall be taken to indicate that a specific integer may be obtained from a particular source albeit not necessarily directly from that source. 15 Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. Other definitions for selected terms used herein may be found within the detailed 20 description of the invention and apply throughout. Unless otherwise defined, all other scientific and technical terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the invention belongs. Examples 25 The following methods serve to more fully describe the manner of using the above-described invention, as well as to set forth the best modes contemplated for carrying out various aspects of the invention. It is understood that these methods in no way serve to limit the true scope of this invention, but rather are presented for illustrative purposes. 0I ID TITI IT" LII""T IDI II I"" -)&% OftiA I I WO 2008/141358 PCT/AU2008/000614 -14 Example I Materials/methods Trial 1: Samples of raw fish from four fish species were investigated, Pearl Perch (Glaucosoma spp); Dhufish (Glaucosoma hebraicum); Nile Perch (Lates 5 niloticus) and Redspot Emperor. Trail 2: Samples of cooked fish from four fish species were investigated, Pearl Perch (Glaucosoma spp); Dhufish (Glaucosoma hebraicum); Nile Perch (Lates niloticus) and Redspot Emperor. Trial 3: Samples of raw fish from Barramudi (Lates calcarifer) were investigated. 10 Trial 4: Samples of raw fish from a further five fish species, Sand whiting (Sillago ciliata), Leather jacket (Meuschenia spp), Tropical snapper (Lutanus vitta), Red Mullet (Upeneus tragula), and Frypan bream (Argyrops spinifer) were then analysed using the same methodology. Trial 5: Samples of raw shark, from Gummy shark (Mustelus antarcticus) and 15 Dusky shark (Carcharhinus obscures) were analysed using the same methodology Trial 6: The methodology was also used to analyse four varieties of wheat (Dagger, Calingiri, Arrino, and Frame) Trial 7: The methodology was also used to analyse four varieties of subclover 20 (Dalkeith, York, Larisa, and Trikkala). Trial 1 involved steps (a)1-5 of the following sample preparation method. Trial 2 involved all steps of the following sample preparation method with samples of cooked fish being first microwaved on High for 5 min to cook thoroughly prior to mincing. Trials 3-7 involved steps 1-5 and step 7 of the following sample 25 preparation and analysis methods. 0I IDOTITI IT"" OLII""T IDI II " -)&% OftiA I I WO 2008/141358 PCT/AU2008/000614 - 15 (a) Sample Preparation 1. Take 2 g of raw tissue (fresh or frozen) and mince roughly with scalpel blades 2. Transfer tissue to a plastic tube and add 10 ml of ice-cold water 3. Mince finely with a mechanical homogeniser for 5 min 5 4. Transfer the homogenate into plastic tubes and centrifuge at 14,000-16,000 x g for 30 min at 40C 5. Recover supernatant (aliquots of supernatant can be stored at -20 (C) 6. To 1 ml supernatant, add 2pl 0.05%(v/v) n-Decy/-NN-Dimethy/-3-Ammonio 1-Propane-Sulfonate (sulphobetaine 10 or SB 3-10) and mix thoroughly 10 other mild surfactants may be used instead. N.B This step may be omitted 7. Transfer the mixture to a Vivaspin-2 10 kDa cutoff CTA-membrane spin concentrator, and spin at 4,000 x g for 30 min at RT (aliquots of supernatant can be stored at -20 0C) 8. N.B This step is omitted if step 6 is omitted. Prewash a BioRad Biospin-6 15 desalting column by: i. removing cap and snapping off base seal ii. draining column under gravity iii. performing three 1 ml water washes, each time draining under gravity iv. after final wash, spin column for 2 min at 1,000 x g and discard eluate 20 9. N.B This step is omitted if step 6 is omitted. Load washed column with 75 pil of the supernatant from step 7 10.N.B This step is omitted if step 6 is omitted. Spin at 1,000 x g for 4 min (aliquots of eluate can be stored at -20 OC) (b) Analysis of Protein Subset by Mass Spectrometry 25 1. Dry 30 pl of fish/wheat/subclover protein extract (<10 kDa) from above by rotary vacuum evaporation 2. Add 10 pi of digest solution (12.5 pg/ml trypsin) and incubate at 37 "C overnight I ID TITI IT"" OLII""T IDI II " ')tl D/"iA I I WO 2008/141358 PCT/AU2008/000614 -16 3. Dry the sample by rotary vacuum evaporation 4. (A) Perform analysis on a 4800 MALDI TOF/TOF mass spectrometer (Applied Biosytems) controlled by GPS Explorer software (Applied Biosytems), OR (B) Perform on-line liquid chromatography/mass spectrometry (LC/MS) analysis 5 of dissolved sample using two series 1100 LC Pumps (Agilent) connected to the PE-SCIEX ionspray source of a QSTAR spectrometer (Applied Biosytems), controlled by Analyst QS software (Applied Biosytems) 5. For 5(B) only: Fractionate the sample using an analytical C18 column with a gradient formed by mixing solvent A (0.1% formic acid) and solvent B (100% 10 acetonitrile in 0.1% formic acid) 6. Analyse the sample in positive ion mode 7. Obtain total ion list from sample and select charged species for further analysis (n=5-200+) 8. Compare at least the top (by intensity) 50 ions, or compare all ions present in 15 the sample (c) Electrophoresis Fish protein samples were electrophoresed on a 10-20% acrylamide BioRad Criterion pre-cast polyacrylamide gel in the presence of sodium dodecy sulfate (SDS) and stained with Coomassie Brilliant Blue G250. 20 Results (a) Analysis of extracts of raw and cooked fish samples The extracts from raw and cooked fish tissue (prior to spin filtration step) from Trials I and 2 were electrophoresed on a 10-20% acrylamide BioRad Criterion pre-cast polyacrylamide gel in the presence of sodium dodecyl sulfate (SDS) and 25 stained with Coomassie Brilliant Blue G250. The results are depicted in Figure 1. The cooked fish tissue from Trial 2, after spin filtration, were electrophoresed on a. 10-20% acrylamide BioRad Criterion pre-cast polyacrylamide gel in the 0I ID TITI IT" LII"""T IDI II I"" -)&% OftiA I I WO 2008/141358 PCT/AU2008/000614 -17 presence of sodium dodecyl sulfate (SDS) and stained with Coomassie Brilliant Blue G250 and are depicted in Figure 2. (b) Analysis of extracts of raw fish samples and wheat/subclover samples following Mass Spectrometry 5 Mass tolerance of instruments The mass tolerance of the Q-Star LC/MS instrument was evaluated using a standard protein. Bovine serum albumin (BSA) was trypsin digested and run on 22 different days. From the spectra of the BSA digest, three selected peaks across the mass range were used to evaluate mass tolerance. The mass 10 tolerance for this set of data is +0.1. The mass tolerance of the MALDI TOF/TOF instrument was also evaluated using a standard trypsin digested BSA protein, with 29 samples run on 16 different days. From the spectra of the BSA digest, 4 selected peaks across the mass range were used to evaluate mass tolerance. The mass tolerance for trials 6 and 15 7 was set at +0.1, whilst that of trials 3, 4 and 5 was set at +0.2. Sample Analysis For Trial 3 (Barramundi), extracts were taken from each of nine individuals, giving a total of 9 extracts. For Trial 4 (five further fish species), one extract was taken from each of four individuals of each species for Q-Star analysis and one 20 extract was taken from each of six individuals for MALDI analysis. For Trial 5 (two shark species) extracts were taken from 7 Gummy shark individuals and 10 Dusky shark individuals. For the wheat and subclover of Trials 6 and 7, three individuals per variety were sampled and one extract was taken from each individual. 25 All protein extracts from each species or variety were run on a mass spectrometer (either Q-Star LC/MS or MALDI or both) and analysed. For Q-Star 0I IDOTITI IT"" OLII"""T IDI II " )&% f"IA I I WO 2008/141358 PCT/AU2008/000614 - 18 analysis, all ions present were compared. For MALDI profiles, the 150 ions with the strongest signal were selected for comparison between samples. The master mass spectrometry profiles were compared between extracts. Present/non-present peptide ion analysis was performed and omnipresent 5 peptide ions were identified. All the samples of proteins obtained from a given sample type (eg fish, shark, wheat, subclover etc) were first compared to identify ions that were present in all the samples, regardless of species. Thus, the first set of peptide ions identified was the set of ions that occurred in all species of, for example, fish. This set of 10 peptide ions identified could be used to determine that a given protein sample was obtained from fish. From Trial 4 and Figure 4 it can be seen that Q-Star analysis resulted in 27 peptide ions and MALDI analysis resulted in 3 peptide ions which were present in all samples of fish, regardless of species, and which could be used to identify a protein sample as deriving from fish. Similarly, a set 15 of seven peptides were identified in all wheat samples, which forms a signature indicating that a given protein sample derived from wheat. A second set of peptide markers was identified which indicated the species or variety of origin of a given protein sample. A number of samples were obtained from different members of the same species or variety, and the peptide markers 20 compared. Once the common markers that identified the type of sample (eg fish, shark, wheat, subclover) were identified, the remaining ions that were present in all samples from a given species or variety formed a peptide profile that could be used to identify the species from which the sample came. Table 1 below shows the identified peptide ions for each species. 25 0I ID TITIIT"" OLI""T IDIII I"" )&% f"IA II WO 2008/141358 PCT/AU2008/000614 - 19 Table 1 Signature Forming Peptide Biomarkers LC/MS MALDI Fish Barramundi 7 9 Sand Whiting 18 34 Leather Jacket 7 30 Tropical Snapper 11 53 Red Mullet 19 32 Frypan bream 24 37 Shark Gummy shark 10 Dusky shark 13 Wheat Dagger 5 Caligiri 29 Arrino 7 Frame 21 Subclover Dalkeith 25 York 18 Larisa 10 Trikkala 9 From these results, a number of peptide markers were established for each sample species. For example, for Barramundi, seven peptide markers were 5 established using Q-Star analysis, and nine peptide markers were established using MALDI analysis, forming a peptide signature. Thus, samples which displayed a profile with that peptide signature (i.e. those seven Q-Star peptide markers or 9 MALDI peptide markers) could be positively identified as deriving from Barramundi. For Sand whiting, a peptide signature of 18 peptide markers 10 generated by Q-Star or a peptide signature of 34 peptide markers generated by MALDI could be used to positively identify the source of a sample as being Sand whiting. For Gummy shark, a peptide signature of 10 peptide markers generated by MALDI could be used to establish that the source of a sample was Gummy shark. 15 Figures 3 to 7 are flow charts detailing the workflow and results of experiments with raw fish samples, shark samples and wheat and subclover samples. 0I IDOTITI IT"" OLII""T IDI II " -)&% OftiA I I WO 2008/141358 PCT/AU2008/000614 - 20 Modifications of the above-described modes of carrying out the various embodiments of this invention will be apparent to those skilled in the art based on the above teachings related to the disclosed invention. The above embodiments of the invention are merely exemplary and should not be construed 5 to be in any way limiting. 0I IDOTITI ITC LICCT IDI II C -)&% OftiA I I

Claims (21)

1. A method for determining the source of a protein sample comprising the steps of: (i) isolating a subset of proteins from the sample; 5 (ii) fragmenting one or more of the proteins in the protein subset; (iii) characterising the proteins and protein fragments of the protein subset; and (iv) using the results from step (iii) to determine the origin of the sample.

2. The method of claim 1 wherein the protein sample is from a food source. 10 3. The method of claim 1 wherein the food source is a fungi, plant or animal tissue.

4. The method of claim 3 wherein the animal tissue is meat from livestock which are used as food for animals and/or humans or wherein the animal tissue is from a fish selected from the group comprising: Lethrinus spp including L. 15 lentjan, Lates spp including L. niloticus and L. calcarifer, Glaucosoma spp including G. hebraicum, G. scapulare, G. buergeri, G magnificans, Plectropoums spp such as P. leopardus and members of the Merluciidae family such as various Merluccius spp.

5. The method of claim 1 wherein the proteins are extracted from the sample 20 prior to isolation.

6. The method of claim 5 wherein the extraction is carried out using a surfactant.

7. The method of claim 6 wherein the surfactant is chosen from the group comprising: zwitterionic detergents such as n-Decyl-N,N-Dimethyl-3 25 Ammonio-1-Propane-Sulfonate (sulphobetaine 10 or SB 3-10) or 3-[(3 OI IDOTITI ITC OLICCT IDI II C n&% Of"IA I I WO 2008/141358 PCT/AU2008/000614 -22 Cholamidopropyl)dimethylammonio]-1 -propanesulfonate (CHAPS); 3 (Dodecyldimethylammonio)propanesulfonate; 3-(Dodecyldimethylammonio) propanesulfonate; 3-(N,N-Dimethylmyristylammonio)propanesulfonate; 3 (N,N-Dimethyloctadecylammonio)propanesulfonate; 3-(N,N 5 Dimethyloctylammonio) propanesulfonate; and 3-(N,N Dimethylpalmitylammonio)propanesulfonate.

8. The method of claim 6 wherein the amount of surfactant used to extract the proteins is between 0.01%v/v and 1.0%v/v; between 0.04%v/v and 0.3%v/v or wherein the amount of surfactant is 0.05%v/v. 10 9. The method of claim 1 wherein the subset is isolated by relying on at least one of the following characteristics: size, solubility, charge, isoelectric point, affinity. 1O.The method of claim 9 wherein the subset is isolated using at least size as the defining characteristic. 15 11.The method of claim 9 wherein the subset is isolated by relying on at least two of the characteristics.

12.The method of claim 1 wherein, following isolation, the subset comprises at least 80%, 85%, 90%, 95% or 99% of each of the subset proteins that were in the sample prior to isolation. 20 13.The method of claim 1 wherein the subset is formed of all the proteins in a sample that have a molecular mass less than 1OkDa.

14.The method of claim 1 wherein the isolation by size is carried out by ultrafiltration.

15.The method of claim 14 wherein the ultrafiltration is selected from the list 25 comprising: centrifugal filtration with pressure, centrifugal filtration without pressure, gas pressure filtration, tangential flow filtration, chromatographic (gel) filtration, and solvent absorption. 0I IDOTITI ITI"" OLII""""T IDI II " n&% OftiA I I WO 2008/141358 PCT/AU2008/000614 -23

16.The method of claim 10 wherein the isolation by size is carried out using mass column filters or spin columns.

17.The method of claim 1 wherein the subset is characterised by forming a profile of the proteins and protein fragments in the subset. 5 18.The method of claim I wherein enzymes are used to treat the proteins and cause fragmentation.

19.The method of claim 18 wherein the enzyme used to treat the proteins and cause fragmentation is trypsin.

20. The method of claim 17 wherein the subset is characterised by one or more 10 techniques selected from the group consisting of: chromatography, for example high-performance liquid chromatography (HPLC), gas chromatography (GC), liquid chromatography (LC), mass spectrometry (MS); electrophoresis, for example 1 D or 2D gels; mass spectrometry using matrix assisted laser desorption ionisation (MALDI); mass spectrometry using 15 electrospray.

21.The method of claim 20 wherein the subset is characterised using mass spectrometry.

22.A method for determining the source of a protein sample comprising the steps of: 20 (i) isolating a subset of proteins from the sample; (ii) fragmenting one or more of the proteins in the protein subset; (iii) characterising the proteins and protein fragments using mass spectrometry; and (iv) using the results from step (iii) to determine the origin of the sample. 0I IDOTITI IT"" OLI"T IDI II I"" -)&% OftiA I I WO 2008/141358 PCT/AU2008/000614 -24

23.The method of claim 22 wherein the results are compared to a database containing at least one reference result indicative of an identity.

24.A method for determining the source of a protein sample comprising the steps of: 5 (i) isolating a subset of proteins from the sample; (ii) fragmenting one or more of the proteins in the protein subset; (iii) characterising the proteins and protein fragments using mass spectrometry; (iv) using the results from step (iii) to prepare a peptide signature; and 10 (v) using the peptide signature from step (iv) to determine the origin of the sample.

25.The method of claim 24 wherein the signature prepared in step (iii) has less than 50 peaks and even more preferably about 10 peaks.

26.The method of claim 24 comprising the further step of sequencing the 15 peptides in the signature.

27.A method for preparing a database of peptide signatures comprising the steps of: (i) isolating a subset of proteins from a protein sample; (ii) fragmenting one or more of the proteins in the protein subset; 20 (iii) characterising the proteins and protein fragments; and (iv) placing the results from step (iii) into a database. CI IID TITI IT I LII"""T IIE I I '%I" " ^ IIA I I