The objectives of the current study were to profile changes in protein composition using 2-dimensional gel electrophoresis on whey samples from a group of 8 cows before and 18 h after infection with Escherichia coli and to identify differentially expressed milk proteins by peptide sequencing using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry post source decay. Only proteins present in whey fractions of all 8 cows were sequenced to avoid reporting a protein response unique to only a subset of infected cows. Despite the overwhelming presence of casein and beta-lactoglobulin, the low abundance proteins transthyretin, lactadherin, beta-2-microglobulin precursor, alpha-1-acid glycoprotein, and complement C3 precursor could be identified in whey samples from healthy cows. Whey samples at 18 h postinfection were characterized by an abundance of serum albumin, in spots of varying mass and isoelectric point, as well as increased transthyretin and complement C3 precursor levels. Also detected at 18 h postinoculation were the antimicrobial peptides cathelicidin, indolicidin, and bactenecin 5 and 7, and the proteins beta-fibrinogen, alpha-2-HS-glycoprotein, S100-A12, and alpha-1-antiproteinase. Most notable was the detection of the acute phase protein alpha-1-acid glycoprotein in mastitic whey samples, a result not previously reported. In contrast to methods used in previous proteomic analyses of bovine milk, the methods used in the current study enabled the rapid identification of milk proteins with minimal sample preparation. Use of a larger sample size than previous analyses also allowed for more robust protein identification. Results indicate that examination of the protein profile of whey samples from cows after inoculation with E. coli could provide a rapid survey of milk protein modulation during coliform mastitis and aid in the identification of biomarkers of this disease.

Detection of peptides from the peanut allergen Ara h 1 by liquid chromatography-mass spectrometry (LC-MS) was used to identify and estimate total peanut protein levels in dark chocolate. A comparison of enzymatic digestion subsequent to and following extraction of Ara h 1 from the food matrix revealed better limits of detection (LOD) for the pre-extraction digestion (20 ppm) than for the postextraction digestion (50 ppm). Evaluation of LC-MS instruments and scan modes showed the LOD could be further reduced to 10 ppm via a triple-quadrupole and multiple-reaction monitoring. Improvements in extraction techniques combined with an increase in the amount of chocolate extracted (1 g) improved the LOD to 2 ppm of peanut protein. This method provides an unambiguous means of confirming the presence of the peanut protein in foods using peptide markers from a major allergen, Ara h 1, and can easily be modified to detect other food allergens.

Enzymatic digestion of total protein along with liquid chromatography/tandem mass spectrometry (LC/MS/MS) was used to confirm the presence of a major peanut allergen in food. Several peptides obtained from the enzymatic digestion of the most abundant peanut allergen, Ara h 1, were identified as specific peptide biomarkers for peanut protein. Using ice cream as a model food matrix, a method was developed for the detection of the allergen peptide biomarkers. A key component of the method was the use of molecular mass cutoff filters to enrich the Ara h 1 in the protein extracts. By applying the method to ice cream samples containing various levels of peanut protein, levels as low as 10 mg/kg of Ara h 1 could routinely be detected. This method provides an unambiguous means of confirming the presence of the peanut allergen, Ara h 1, in foods and can easily be modified to detect other food allergens.

Although immunoassay-based methods are sensitive and widely used for measuring protein toxins in food matrixes, there is a need for methods that can directly confirm the molecular identity of the toxin in situations where immunoassay tests yield a positive result. A method has been developed that uses mass spectrometry to identify a protein toxin, staphylococcal enterotoxin B (SEB), in a model food matrix, apple juice. The approach employs ultrafiltration to remove low molecular weight components from the sample, after which the remaining high molecular weight fraction, containing the protein, is digested with trypsin. The tryptic fragments are separated from residual biopolymers and analyzed by liquid chromatography-electrospray mass spectrometry. The background is still sufficiently complex that tandem mass spectrometry (MS/MS) is used to confirm the identity of target peptides. Limits of detection are 80 ng of SEB for MS and 100 ng for full scan MS/MS, using a tryptic fragment as the analytical target. Lower detection limits can be obtained using selected ion monitoring and multiple reaction monitoring. The presence of SEB can be confirmed at concentrations as low as 5 parts-per-billion by increasing the size of the sample to 10 mL. The method is applicable to the detection of SEB in other water-soluble food matrixes.