Milk Bottom-Up Proteomics: Method Optimization

Abstract

Milk is a complex fluid whose proteome displays a diverse set of proteins of high abundance such as caseins and medium to low abundance whey proteins such as ß-lactoglobulin, lactoferrin, immunoglobulins, glycoproteins, peptide hormones, and enzymes. A sample preparation method that enables high reproducibility and throughput is key in reliably identifying proteins present or proteins responding to conditions such as a diet, health or genetics. Using skim milk samples from Jersey and Holstein-Friesian cows, we compared three extraction procedures which have not previously been applied to samples of cows' milk. Method A (urea) involved a simple dilution of the milk in a urea-based buffer, method B (TCA/acetone) involved a trichloroacetic acid (TCA)/acetone precipitation, and method C (methanol/chloroform) involved a tri-phasic partition method in chloroform/methanol solution. Protein assays, SDS-PAGE profiling, and trypsin digestion followed by nanoHPLC-electrospray ionization-tandem mass spectrometry (nLC-ESI-MS/MS) analyses were performed to assess their efficiency. Replicates were used at each analytical step (extraction, digestion, injection) to assess reproducibility. Mass spectrometry (MS) data are available via ProteomeXchange with identifier PXD002529. Overall 186 unique accessions, major and minor proteins, were identified with a combination of methods. Method C (methanol/chloroform) yielded the best resolved SDS-patterns and highest protein recovery rates, method A (urea) yielded the greatest number of accessions, and, of the three procedures, method B (TCA/acetone) was the least compatible of all with a wide range of downstream analytical procedures. Our results also highlighted breed differences between the proteins in milk of Jersey and Holstein-Friesian cows.

Proteomics, lipidomics, metabolomics and 16S DNA sequencing of dental plaque from patients with diabetes and periodontal disease

“…The next most closely correlated proteins with these TGs were also assigned to taxonomic family Bovidae (alpha-S2-casein, kappa-casein, and beta-lactoglobulin). Together with APO-A1, these proteins constitute some of the most abundant proteins in cow’s milk 44 . We annotated eight proteins to the taxonomic family Bovidae , and five of the eight showed strong correlations to medium-chain containing TGs ( Figure 6d ).…”

Lactic acid‐mediated isolation of alpha‐, beta‐ and kappa‐casein fractions by isoelectric precipitation coupled with cold extraction from defatted cow milk

“…The stored frozen full cream milk was thawed and defatted by centrifugation at 2060 g at 4 ºC for 30 min. The skim milk portion between the fatty upper layer and lower pellet cellular portion was withdrawn carefully and stored at −80 ºC prior to isolation (Ahmed et al ; Vincent et al ).…”

Top-Down Proteomics of Medicinal Cannabis

“…Standard solutions were then desalted using solid phase extraction (SPE) cartridges (Sep-Pak C18 1cc Vac Cartridge, 50 mg sorbent, 55-105 µm particle size, 1 mL, Waters, Milford, MA, USA) by gravity as described in [30]. Bound intact proteins were desalted using 1 mL of 0.1% FA solution and eluted into a 2 mL microtube using 1 mL of 80% ACN/0.1% FA solution.…”

“…The commercial standards used in this study and past work [28,30] contained mixtures of protein isoforms. Deconvolution of full scan FTMS1 ( Figure 4A) supplied accurate masses for β-lactoglobulin, α-S1-casein, and average masses for BSA with an error <50 ppm, which helped in figuring out which protein isoforms underwent MS/MS analysis and which sequence to use for ProSight Lite annotation.…”

“…This work follows on from our optimization of protein extraction of mature buds from medicinal cannabis prior to bottom-up proteomics [1]. In this top-down study, we used a hybrid mass spectrometer composed of a linear ion trap (LTQ or ITMS) and a Fourier-transform Orbitrap (FTMS), which we typically use for bottom-up experiments [1,30]. This powerful instrument offers high resolution (up to 240,000) and multiple modes of fragmentations (SID, CID, HCD, and ETD) relative to the Q-ToF system employed previously.…”