Volatile fingerprints of beef cooking methods using sol-gel-based solid-phase microextraction (SPME) and direct analysis in real-time mass spectrometry (DART-MS).

RATIONALE: The aroma profile of food is a complex mixture of volatile compounds that constitutes a major component of the overall eating experience. The food service industry and chefs therefore constantly seek ways to investigate and thereby enhance the aroma profile. Oven cooking, sous vide and pan fry are three cooking methods of beef commonly practised by chefs. Near real-time analysis of volatile compounds from these three cooking methods will provide insight into respective volatile fingerprints and help improve cooking techniques. METHODS: Volatile compounds from three beef cooking methods were captured using an in-house sol-gel based solid phase microextraction (SPME) method and analysed using direct analysis in real-time mass spectrometry (DART-MS). A volatile organic compound (VOC) standard was used to demonstrate successful implementation of the sol-gel coating technique. Volatile features discriminating the three cooking methods were shortlisted and statistically assessed by univariate and multivariate analyses. RESULTS: The VOC standard was successfully adsorbed by the sol-gel method and detected by DART-MS. Hierarchical cluster analysis clearly demarcated three beef cooking methods based on their volatile fingerprints. Out of 65 significant features differentiating the cooking methods, 50 were at highest concentrations from pan-fry cooking only, followed by 14 with highest concentrations from oven cooking followed by pan frying. Sous vide followed by pan frying showed lowest concentrations of almost all volatile features. CONCLUSIONS: The sol-gel-based solid-phase microextraction technique combined with DART-MS was successful in differentiating beef cooking methods based on their volatile fingerprints. A workflow for rapid assessment of the volatile profile from beef cooking methods was established, providing a baseline to further explore volatile profiles from other key ingredients.

Molecular insights into quality and authentication of sheep meat from proteomics and metabolomics.

Sheep meat (encompassing lamb, hogget and mutton) is an important source of animal protein in many countries, with a unique flavour and sensory profile compared to other red meats. Flavour, colour and texture are the key quality attributes contributing to consumer liking of sheep meat. Over the last decades, various factors from 'farm to fork', including production system (e.g., age, breed, feeding regimes, sex, pre-slaughter stress, and carcass suspension), post-mortem manipulation and processing (e.g., electrical stimulation, ageing, packaging types, and chilled and frozen storage) have been identified as influencing different aspects of sheep meat quality. However conventional meat-quality assessment tools are not able to elucidate the underlying mechanisms and pathways for quality variations. Advances in broad-based analytical techniques have offered opportunities to obtain deeper insights into the molecular changes of sheep meat which may become biomarkers for specific variations in quality traits and meat authenticity. This review provides an overview on how omics techniques, especially proteomics (including peptidomics) and metabolomics (including lipidomics and volatilomics) are applied to elucidate the variations in sheep meat quality, mainly in loin muscles, focusing on colour, texture and flavour, and as tools for authentication. SIGNIFICANCE: From this review, we observed that attempts have been made to utilise proteomics and metabolomics techniques on sheep meat products for elucidating pathways of quality variations due to various factors. For instance, the improvement of colour stability and tenderness could be associated with the changes to glycolysis, energy metabolism and endogenous antioxidant capacity. Several studies identify proteolysis as being important, but potentially conflicting for quality as the enhanced proteolysis improves tenderness and flavour, while reducing colour stability. The use of multiple analytical methods e.g., lipidomics, metabolomics, and volatilomics, detects a wider range of flavour precursors (including both water and lipid soluble compounds) that underlie the possible pathways for sheep meat flavour evolution. The technological advancement in omics (e.g., direct analysis-mass spectrometry) could make analysis of the proteins, lipids and metabolites in sheep meat routine, as well as enhance the confidence in quality determination and molecular-based assurance of meat authenticity.

Unlocking the bioactivity of meat proteins: Comparison of meat and meat hydrolysate via simulated gastrointestinal digestion.

Understanding the functional attributes of meat proteins is crucial for determining their nutritional benefits. Depending on the form in which meat proteins are available, the digestive process can release peptides which are valuable for nutrition and may also possess bioactive properties, affecting physiology. Liquid chromatography - mass spectrometry (LC-MS) was used to quantitatively compare the molecular peptide features (representing non-redundant peptides), during the different stages of a simulated gastrointestinal digestion process of a minimally processed powdered meat and its enzymatically produced hydrolysate. Results from a principal component analysis (PCA) indicated that the hydrolysate did not undergo extensive additional digestion whereas the powdered meat was digested both at the gastric and in the intestinal phases. Bioactive peptide sequence prediction identified the meat hydrolysate but not the meat powder as the only source of exact and partial bioactive matches in the angiotensin-I converting enzyme and dipeptidyl peptidase IV inhibition categories. Also, a higher source of cryptides (encrypted bioactive peptides), indicated that meat hydrolysates are potentially a better substrate for the release of these enzyme inhibitory peptides. These observations thus suggest that pre-digestion of a complex food matrix such as meat, may enhance its bioavailability following oral consumption early in the digestion process. SIGNIFICANCE: This work highlights enzymatic hydrolysis of meat proteins prior to ingestion allows for potentially higher bioavailability of bioactive peptides that inhibit angiotensin-I converting enzyme and dipeptidyl peptidase IV, thus possibly aiding high blood pressure and type 2 diabetes management.

Generation and identification of kokumi compounds and their validation by taste-receptor assay: An example with dry-cured lamb meat.

Kokumi tastants are small γ-glutamyl peptides (GGP) that enhance flavour in foods. We sought to generate GGP from the meat crusts of dry-cured lamb, an underutilised protein resource, identify these using mass spectrometry, and validate their functional activity using a kokumi-calcium sensing receptor (CaSR) assay. The water-soluble extract (WSE) of meat crust was hydrolysed by protease A (PA) and treated with glutaminase (GA). Fifteen GGP were identified, with 14 being significantly increased in PA and GA groups compared to WSE, as were along with free amino acid levels. The GA extract activated CaSR with higher potency and efficacy than PA and WSE suggesting the generation of potent kokumi tastants. The in vitro receptor assay might be an expedient tool for screening kokumi tastants prior to conducting human sensory analysis. Collectively, our findings indicate that the meat crust can be a valuable source to generate kokumi tastants via a two-step enzymatic reaction.

Proteomic Profile of M. longissimus thoracis from Commercial Lambs Reared in Different Forage Systems.

This study compared the protein composition of M. longissimus thoracis of lambs from six commercial forage production systems in New Zealand. A total of 286 proteins were identified based on liquid chromatography-tandem mass spectrometry. First, a binomial model showed that different production groups could be distinguished based on abundances of 16 proteins. Second, pair-wise comparisons were performed to search for protein abundance differences in meat due to animal sex (ewe vs. wether), diet (perennial ryegrass vs. chicory), and age (4 vs. 6-8 months old). Greater abundance of some myofibrillar and sarcoplasmic proteins were observed in lamb loins from ewes compared to wethers. Chicory diet and older age at slaughter were associated with meat with lower abundance of some myofibrillar proteins, possibly due to a greater proportion of muscle glycolytic fibres. The proteins that showed significant differences in their abundances due to production factors could be further investigated to understand their influence on meat quality.

The susceptibility of disulfide bonds to modification in keratin fibers undergoing tensile stress.

Cysteine residues perform a dual role in mammalian hairs. The majority help stabilize the overall assembly of keratins and their associated proteins, but a proportion of inter-molecular disulfide bonds are assumed to be associated with hair mechanical flexibility. Hair cortical microstructure is hierarchical, with a complex macro-molecular organization resulting in arrays of intermediate filaments at a scale of micrometres. Intermolecular disulfide bonds occur within filaments and between them and the surrounding matrix. Wool fibers provide a good model for studying various contributions of differently situated disulfide bonds to fiber mechanics. Within this context, it is not known if all intermolecular disulfide bonds contribute equally, and, if not, then do the disproportionally involved cysteine residues occur at common locations on proteins? In this study, fibers from Romney sheep were subjected to stretching or to their breaking point under wet or dry conditions to detect, through labeling, disulfide bonds that were broken more often than randomly. We found that some cysteines were labeled more often than randomly and that these vary with fiber water content (water disrupts protein-protein hydrogen bonds). Many of the identified cysteine residues were located close to the terminal ends of keratins (head or tail domains) and keratin-associated proteins. Some cysteines in the head and tail domains of type II keratin K85 were labeled in all experimental conditions. When inter-protein hydrogen bonds were disrupted under wet conditions, disulfide labeling occurred in the head domains of type II keratins, likely affecting keratin-keratin-associated protein interactions, and tail domains of the type I keratins, likely affecting keratin-keratin interactions. In contrast, in dry fibers (containing more protein-protein hydrogen bonding), disulfide labeling was also observed in the central domains of affected keratins. This central "rod" region is associated with keratin-keratin interactions between anti-parallel heterodimers in the tetramer of the intermediate filament.

Redox proteomics analysis of hair shaft proteins upon hydrothermal and alkaline insult.

OBJECTIVE: Human hair is regularly subjected to chemical and physical insults, such as heat, UV-irradiation and alkaline hair care products. These insults result in molecular modifications at the hair protein level that underpin mechanical and sensory property changes in the fibres. These changes can manifest itself in reduced hair quality and performance attributes observable to the consumer. In this work, changes in protein modification as a result of heat and alkaline treatments are determined. METHODS: Redox proteomic profiling using high-resolution mass spectrometry was applied to map and evaluate amino acid residue modifications in human hair exposed to a combination of thermal treatments and alkali exposure with the aim to understand the underlying chemical processes. RESULTS: Our results show that an increase in redox-related modifications is associated with exposure to higher levels of hydrothermal and alkaline insult. Post-translational modification profiling at the protein primary structural level delivered some further insights into the site-specificity of these modifications, with a clear increase in the number of cysteic acid modifications noticed in samples subjected to more extreme insults. CONCLUSION: Pinpointing modification sides within proteins and the hair shaft proteome can be used as a basis for employing mitigation or repair strategies of hair protein damage caused by environmental or hair treatment-related insults.

OBJECTIF: Les cheveux humains sont sujet à de nombreuses agressions physiques et chimiques telles que la chaleur, les radiations ultra-violettes et les produits alcalins d'entretien des cheveux. Ces agressions entrainent des modifications moléculaires dans les protéines constituant les cheveux et elles conduisent aussi à des changements mécaniques et sensoriels des fibres capillaires. Les manifestations possibles de ces transformations sont une baisse, visible pour le consommateur, de la qualité et des indicateurs de performance des cheveux. Lors de cette étude, nous mettons en évidence les changements au niveau protéique liés à la chaleur et aux traitements alcalins. MÉTHODES: Les méthodes de profilage d'oxydoréduction protéomique utilisant des spectromètres de masses à haute résolution ont été utilisées afin d'évaluer les modifications des amino-acides dans les cheveux humains après exposition à plusieurs combinaisons de traitements thermiques et alcalins dans le but de comprendre les processus chimiques impliqués. RÉSULTATS: Nos résultats montrent que l'augmentation des modifications d'oxydoréduction est associée à des niveaux élevés d'exposition aux traitements thermiques et/ou alcalins. Le profilage des modifications post-translationnelles des structures primaires des protéines ont permis de mieux comprendre les spécificités de ces modifications ; notamment une augmentation nette du nombre des modifications des acides cystéiques liée aux traitements les plus agressifs. CONCLUSION: Ce travail d'identification des modifications engendrées par les agressions liées aux traitements capillaires ou environnementales peut désormais servir de base pour évaluer et mettre en place des techniques de réduction des risques, protection et de réparation des protéines des cheveux.

Genomic Tools for the Identification of Loci Associated with Facial Eczema in New Zealand Sheep.

Facial eczema (FE) is a significant metabolic disease that affects New Zealand ruminants. Ingestion of the mycotoxin sporidesmin leads to liver and bile duct damage, which can result in photosensitisation, reduced productivity and death. Strategies used to manage the incidence and severity of the disease include breeding. In sheep, there is considerable genetic variation in the response to FE. A commercial testing program is available for ram breeders who aim to increase tolerance, determined by the concentration of the serum enzyme, gamma-glutamyltransferase 21 days after a measured sporidesmin challenge (GGT21). Genome-wide association studies were carried out to determine regions of the genome associated with GGT21. Two regions on chromosomes 15 and 24 are reported, which explain 5% and 1% of the phenotypic variance in the response to FE, respectively. The region on chromosome 15 contains the ß-globin locus. Of the significant SNPs in the region, one is a missense variant within the haemoglobin subunit ß (HBB) gene. Mass spectrometry of haemoglobin from animals with differing genotypes at this locus indicated that genotypes are associated with different forms of adult ß-globin. Haemoglobin haplotypes have previously been associated with variation in several health-related traits in sheep and warrant further investigation regarding their role in tolerance to FE in sheep. We show a strategic approach to the identification of regions of importance for commercial breeding programs with a combination of discovery, statistical and biological validation. This study highlights the power of using increased density genotyping for the identification of influential genomic regions, combined with subsequent inclusion on lower density genotyping platforms.

A detailed mapping of the readily accessible disulphide bonds in the cortex of wool fibres.

Trichocyte keratin intermediate filament proteins (keratins) and keratin associated proteins (KAPs) differ from their epithelial equivalents by having significantly more cysteine residues. Interactions between these cysteine residues within a mammalian fiber, and the putative regular organization of interactions are likely important for defining fiber mechanical properties, and thus biological functionality of hairs. Here we extend a previous study of cysteine accessibility under different levels of exposure to reducing compounds to detect a greater resolution of statistically non-random interactions between individual residues from keratins and KAPs. We found that most of the cysteines with this non-random accessibility in the KAPs were close to either the N- or C- terminal domains of these proteins. The most accessible non-random cysteines in keratins were present in the head or tail domains, indicating the likely function of cysteine residues in these regions is in readily forming intermolecular bonds with KAPs. Some of the less accessible non-random cysteines in keratins were discovered either close to or within the rod region in positions previously identified in human epithelial keratins as involved in crosslinking between the heterodimers of the tetramer. Our present study therefore provides a deeper understanding of the accessibility of disulfides in both keratins and KAPs and thus proves that there is some specificity to the disulfide bond interactions leading to these inter- and intra-molecular bonds stabilizing the fiber structure. Furthermore, these suggest potential sites of interaction between keratins and KAPs as well as keratin-keratin interactions in the trichocyte intermediate filament.

The effects of blanching on composition and modification of proteins in navy beans (Phaseolus vulgaris).

Blanching is an important process in the preparation of navy beans (Phaseolus vulgaris L.) for canning. We here explore the effect of blanching which can profoundly affect protein composition and introduce protein-primary-level modifications. Amino acid analysis showed significantly decreased protein abundance (58.5%) in blanched beans compared to raw beans. Proteomic analyses revealed a decrease in high molecular weight isoforms of the major storage globulin proteins phaseolin (mean fold-change -3.7) and legumin (mean fold-change -2.5) and concomitant increase in their low molecular weight isoforms (mean fold-change 6.4 and 8.3, respectively). Blanched beans also had decreased abundance of lipoxygenase (mean fold-change -13.1), an enzyme responsible for product spoilage during storage. Increased lysinoalanine (up to 47%) and highly modified protein fragments were found in the processing waters, indicating heat- induced modifications. Correlating these molecular level changes thus provides a basis for evaluating how processing parameters can be modified to increase protein food quality.

Multi-parameter evaluation of the effect of processing conditions on meat protein modification.

Evaluating the interconnecting effects of pH, temperature and time on food proteins is of relevance to food processing, and food functionality. Here we describe a matrix-based approach in which meat proteins were exposed to combinations of these parameters, selected to cover coordinates in a realistic processing space, and analyzed using redox proteomics. Regions within the matrix showing high levels of protein modification were evaluated for oxidative and other modifications. Both pH and temperature, independently, had a significant effect on the oxidative modifications mostly detected in myofibrillar proteins such as myosin and troponin and also collagen. Heat induced pyroglutamic acid formation was exclusively observed in the myofibrillar proteins. Potential interdependencies between pH, temperature and exposure time were evaluated using a 3-way analysis of variance (ANOVA) on protein modification levels to better understand how industry relevant process parameters influence protein quality and function.

The effects of a wool hydrolysate on short-chain fatty acid production and fecal microbial composition in the domestic cat (Felis catus).

Novel animal-derived fibers are of interest for the pet food industry. We here introduce a method for extracting wool proteins using controlled hydrolysis of wool. This results in an appropriate form and we demonstrate its application in pet food using the domestic cat. The effect of the wool hydrolysate on biomarkers of digestive health (e.g., fecal short-chain fatty acids and fecal microbial composition, apparent amino acid (AA) and protein digestibility), are also described. In a feeding study, a cohort of cats (n = 8 per treatment) were fed a basal diet (Control), or the basal diet supplemented with 2% wool hydrolysate, 2% inulin (Synergy1; as is) or 2% cellulose (Novagel; as is). The concentration of butyric acid was not significant (P = 0.102) between treatment groups. The concentration of fecal lactic acid was greatest (P = 0.007) in cats on the Novagel diet. Valeric acid was increased (P = 0.001) in cats fed Synergy1. Supplementation of cat diet with a wool hydrolysate showed similarities to Novagel supplementation in terms of its effects on fecal short-chain fatty acid concentrations and fecal microbiota composition. Wool hydrolysate increased apparent cysteine digestibility compared to Synergy 1 or Novogel. In terms of fecal health, intake, and palatability, the diet supplemented with wool hydrolysate was not detrimental, being similar to currently used dietary fiber supplements. These findings indicate that wool hydrolysates offer promise as an animal-derived supplement source for pet diets.

Crosslinking Between Trichocyte Keratins and Keratin Associated Proteins.

Trichocyte keratins differ considerably from their epithelial cousins in having a higher number of cysteine residues, of which the greater proportion are located in the head and tail regions of these proteins. Coupled with this is the presence of a large number of keratin associated proteins in these fibres that are high in their cysteine content, the high sulfur proteins and ultra-high sulfur proteins. Thus it is the crosslinking that occurs between the cysteines in the keratins and KAPs that is an important determinant in the functionality of wool and hair fibres. Studies have shown the majority of the cysteine residues are involved in internal crosslinking in the KAPs leaving only a few specific cysteines to interact with the keratins, with most evidence pointing to interactions between these KAP cysteines and the keratin head groups.

Improved Detection and Fragmentation of Disulphide-Linked Peptides.

Characterisation of peptides containing intact disulphide bonds (DSBs) via mass spectrometry is challenging. Our study demonstrates that the addition of aniline to alpha-cyano-4-hydroxycinnamic acid improves detection and fragmentation of complex DSB peptides by matrix-assisted laser desorption/ionization, tandem time-of-flight mass spectrometry (MALDI-TOF-TOF MS). This improved assignment will be a significant new tool when a simple screening to confirm the DSB existence is required.

Protein-protein cross-linking and human health: the challenge of elucidating with mass spectrometry.

INTRODUCTION: In several biomedical research fields, the cross-linking of peptides and proteins has an important impact on health and wellbeing. It is therefore of crucial importance to study this class of post-translational modifications in detail. The huge potential of mass spectrometric technologies in the mapping of these protein-protein cross-links is however overshadowed by the challenges that the field has to overcome. Areas covered: In this review, we summarize the different pitfalls and challenges that the protein-protein cross-linking field is confronted with when using mass spectrometry approaches. We additionally focus on native disulfide bridges as an example and provide some examples of cross-links that are important in the biomedical field. Expert commentary: The current flow of methodological improvements, mainly from the chemical cross-linking field, has delivered a significant contribution to deciphering native and insult-induced cross-links. Although an automated data analysis of proteome-wide peptide cross-linking is currently only possible in chemical cross-linking experiments, the field is well on the way towards a more automated analysis of native and insult-induced cross-links in raw mass spectrometry data that will boost its potential in biomedical applications.

Proteomic and peptidomic differences and similarities between four muscle types from New Zealand raised Angus steers.

Four muscles from New Zealand-raised Angus steers were evaluated (musculus semitendinosus, m. longissimus thoracis et lumborum, m. psoas major and m. infraspinatus) to test their differences and common features in protein and peptide abundances. The ultimate goal of such a comparison is to match muscle types to products with targeted properties. Protein profiling based on two-dimensional electrophoresis showed that the overall profiles were similar, but, between muscle types, significant (p<0.05) intensity differences were observed in twenty four protein spots. Profiling of endogenous peptides allowed characterisation of 346 peptides. Quantitative analysis showed a clear distinction between the muscle types. Forty-four peptides were identified that showed a statistically significant (p<0.05) and substantial (>2-fold change) difference between at least two muscle types. These analyses demonstrate substantial similarities between these four muscle types, but also clear distinctions in their profiles; specifically a 25% difference between at least two muscles at the peptidomic level, and a 14% difference at the proteomic level.

Proteomic tracking of hydrothermal Maillard and redox modification in lactoferrin and ß-lactoglobulin: Location of lactosylation, carboxymethylation, and oxidation sites.

Lactoferrin and ß-lactoglobulin are important protein components of mammalian milk. Maillard reactions, as well as redox chemistry, are of particular interest for dairy products because they are known to occur during common processing steps, notably heating procedures such as pasteurization. Using a redox proteomics approach, we characterized AA residue side-chain modification across a range of heating times and with or without the specific addition of lactose, to both map the key modification sites within these proteins and evaluate their sensitivity to process-induced modification. Heating in the presence of lactose resulted in significant Maillard modification (both lactosylation and carboxymethylation) to both bovine lactoferrin and ß-lactoglobulin. Notably, Lys47, a key residue in the bioactive peptide lactoferricin, was particularly susceptible to modification. Lactoferrin appeared to be fairly robust to hydrothermal treatment, with relatively low levels of oxidative modification observed. In contrast, ß-lactoglobulin was susceptible to significant oxidative modification under hydrothermal treatment, with the range and type of modifications observed suggesting compromised nutritional value. These results have important implications for processing applications in dairy foods where retention of biological function and optimal protein quality is desired.

Application of a Mass Spectrometric Approach to Detect the Presence of Fatty Acid Biosynthetic Phosphopeptides.

The details of plant lipid metabolism are relatively well known but the regulation of fatty acid production at the protein level is still not understood. Hence this study explores the importance of phosphorylation as a mechanism to control the activity of fatty acid biosynthetic enzymes using low and high oleic acid mesocarps of oil palm fruit (Elaeis guineensis variety of Tenera). Adaptation of neutral loss-triggered tandem mass spectrometry and selected reaction monitoring to detect the neutral loss of phosphoric acid successfully found several phosphoamino acid-containing peptides. These peptides corresponded to the peptides from acetyl-CoA carboxylase and 3-enoyl-acyl carrier protein reductase as identified by their precursor ion masses. These findings suggest that these enzymes were phosphorylated at 20th week after anthesis. Phosphorylation could have reduce their activities towards the end of fatty acid biosynthesis at ripening stage. Implication of phosphorylation in the regulation of fatty acid biosynthesis at protein level has never been reported.

Trace metal ions in hair from frequent hair dyers in China and the associated effects on photo-oxidative damage.

Human hairs are subject to oxidative modification when exposed to sunlight. In the present study, samples of human hair from Chinese volunteers that included frequent hair dyers and non-dyers were analyzed for metal ions such as iron, copper, magnesium, aluminum, zinc and lead. The generation of hydroxyl radicals during UVA (315-400 nm) photoageing was quantified and oxidative damages characterized by proteomic and SEM analysis. It was concluded that high levels of metal ions, particularly those derived from iron and copper, identified in the dyed hairs are associated with enhanced photoformation of hydroxyl radicals and resultant photooxidative damage of the hair. Reactive oxygen species, including hydroxyl radicals, generated via an electron transfer mechanism with hair photosensitizers react with hair proteins. Proteomic analysis of hair samples from frequent hair dyers, regardless of age and gender, showed an almost 1.6 fold increase in the protein oxidative modification levels compared to the undyed samples. As a result, a more pronounced physical damage including fragmentation and cross-linkage of cuticle scales was observed on the surface of dyed hair samples during the photoageing. This work is aimed at better understanding the role of metal ions in dyed hairs and their possible role in photosensitizing hair proteins. The results from this study are anticipated to contribute to the improved development of hair coloring cosmetics and hair care products.

Isolation and Analysis of Keratins and Keratin-Associated Proteins from Hair and Wool.

The presence of highly cross-linked protein networks in hair and wool makes them very difficult substrates for protein extraction, a prerequisite for further protein analysis and characterization. It is therefore imperative that these cross-links formed by disulfide bridges are first disrupted for the efficient extraction of proteins. Chaotropes such as urea are commonly used as efficient extractants. However, a combination of urea and thiourea not only improves recovery of proteins but also results in improved resolution of the keratins in 2DE gels. Reductants also play an important role in protein dissolution. Dithiothreitol effectively removes keratinous material from the cortex, whereas phosphines, like Tris(2-carboxyethyl)phosphine, remove material from the exocuticle. The relative extractability of the keratins and keratin-associated proteins is also dependent on the concentration of chaotropes, reductants, and pH, thus providing a means to preferentially extract these proteins. Ionic liquids such as 1-butyl-3-methylimidazolium chloride (BMIM(+)[Cl](-)) are known to solubilize wool by disrupting noncovalent interactions, specifically intermolecular hydrogen bonds. BMIM(+)[Cl](-) proved to be an effective extractant of wool proteins and complementary in nature to chaotropes such as urea and thiourea for identifying unique peptides of wool proteins using mass spectrometry (MS). Successful identification of proteins resolved by one- or two-dimensional electrophoresis and MS is highly dependent on the optimal recovery of its protease-digested peptides with an efficient removal of interfering substances. The detergent sodium deoxycholate used in conjunction with Empore™ disks improved identification of proteins by mass spectrometry leading to higher percentage sequence coverage, identification of unique peptides and higher score.