First Insight into the Variation of the Milk Serum Proteome within and between Individual Cows

Milk contains all nutrients needed for development of calves. One important group of components responsible for this are the milk proteins. Variation due to feed or animal health, has been studied for the most abundant milk proteins. The aim of this study was to determine the variation between and within cows for their milk serum proteome. Sample Set 1 was collected from Holstein Friesian (HF) cows between November 2011 and March 2012 and prepared using filter aided sample preparation (FASP) followed by LC-MS/MS for protein identification and quantification. The results showed that the milk serum proteome was very constant in mid lactation (four cows at five time points, p > 0.05) between 3 and 6 months in lactation. Sample Set 2 was collected from HF cows in Dec 2012 and analyzed using FASP and dimethyl labeling followed by LC-MS/MS. Significant variation in the milk serum proteome (p < 0.05) between 17 individual cows was found in Sample Set 2. The most variable proteins were immune-related proteins, which may reflect the health status of the individual cow. On the other hand, proteins related to nutrient synthesis and transport were relatively constant, indicating the importance of milk in providing a stable supply of nutrients to the neonate. In conclusion, the milk serum proteome was stable over mid lactation, but differed significantly between individuals, especially in immune-related proteins.

Isotopically Dimethyl Labeling-Based Quantitative Proteomic Analysis of Phosphoproteomes of Soybean Cultivars

Isotopically dimethyl labeling was applied in a quantitative post-translational modification (PTM) proteomic study of phosphoproteomic changes in the drought responses of two contrasting soybean cultivars. A total of 9457 phosphopeptides were identified subsequently, corresponding to 4571 phosphoprotein groups and 3889 leading phosphoproteins, which contained nine kinase families consisting of 279 kinases. These phosphoproteins contained a total of 8087 phosphosites, 6106 of which were newly identified and constituted 54% of the current soybean phosphosite repository. These phosphosites were converted into the highly conserved kinase docking sites by bioinformatics analysis, which predicted six kinase families that matched with those newly found nine kinase families. The overly post-translationally modified proteins (OPP) occupies 2.1% of these leading phosphoproteins. Most of these OPPs are photoreceptors, mRNA-, histone-, and phospholipid-binding proteins, as well as protein kinase/phosphatases. The subgroup population distribution of phosphoproteins over the number of phosphosites of phosphoproteins follows the exponential decay law, Y = 4.13e−0.098X − 0.04. Out of 218 significantly regulated unique phosphopeptide groups, 188 phosphoproteins were regulated by the drought-tolerant cultivar under the water loss condition. These significantly regulated phosphoproteins (SRP) are mainly enriched in the biological functions of water transport and deprivation, methionine metabolic processes, photosynthesis/light reaction, and response to cadmium ion, osmotic stress, and ABA response. Seventeen and 15 SRPs are protein kinases/phosphatases and transcription factors, respectively. Bioinformatics analysis again revealed that three members of the calcium dependent protein kinase family (CAMK family), GmSRK2I, GmCIPK25, and GmAKINβ1 kinases, constitute a phosphor-relay-mediated signal transduction network, regulating ion channel activities and many nuclear events in this drought-tolerant cultivar, which presumably contributes to the development of the soybean drought tolerance under water deprivation process.

LC-MS Quantification of Site-Specific Phosphorylation Degree by Stable-Isotope Dimethyl Labeling Coupled with Phosphatase Dephosphorylation

Protein phosphorylation is a crucial post-translational modification that plays an important role in the regulation of cellular signaling processes. Site-specific quantitation of phosphorylation levels can help decipher the physiological functions of phosphorylation modifications under diverse physiological statuses. However, quantitative analysis of protein phosphorylation degrees is still a challenging task due to its dynamic nature and the lack of an internal standard simultaneously available for the samples differently prepared for various phosphorylation extents. In this study, stable-isotope dimethyl labeling coupled with phosphatase dephosphorylation (DM + deP) was tried to determine the site-specific degrees of phosphorylation in proteins. Firstly, quantitation accuracy of the (DM + deP) approach was confirmed using synthetic peptides of various simulated phosphorylation degrees. Afterwards, it was applied to evaluate the phosphorylation stoichiometry of milk caseins. The phosphorylation degree of Ser130 on α-S1-casein was also validated by absolute quantification with the corresponding synthetic phosphorylated and nonphosphorylated peptides under a selected reaction monitoring (SRM) mode. Moreover, this (DM + deP) method was used to detect the phosphorylation degree change of Ser82 on the Hsp27 protein of HepG2 cells caused by tert-butyl hydroperoxide (t-BHP) treatment. The results showed that the absolute phosphorylation degree obtained from the (DM + deP) approach was comparable with the relative quantitation resulting from stable-isotope dimethyl labeling coupled with TiO2 enrichment. This study suggested that the (DM + deP) approach is promising for absolute quantification of site-specific degrees of phosphorylation in proteins, and it may provide more convincing information than the relative quantification method.

A Chemoenzymatic Method for Glycoproteomic N-glycan Type Quantitation

ABSTRACTGlycosylation is one of the most important post-translational modifications in biological systems. Current glycoproteome methods mainly focus on qualitative identification of glycosylation sites or intact glycopeptides. However, the systematic quantitation of glycoproteins has remained largely unexplored. Here, we developed a chemoenzymatic method to quantitatively investigate N-glycoproteome based on the N-glycan types. Taking advantage of the specificity of different endoglycosidases and isotope dimethyl labeling, six N-glycan types of structures linked on each glycopeptide, including high-mannose/hybrid, bi-antennary and tri-antennary with/without core fucose, were quantified. As a proof of principle, the glycoproteomic N-glycan type quantitative (glyco-TQ) method was first used to determine the N-glycan type composition of immunoglobulin G1 (IgG1) Fc fragment. Then we applied the method to analyze the glycan type profile of proteins in the breast cancer cell line MCF7, and quantitatively revealed the N-glycan type micro-heterogeneity at both the glycopeptide and glycoprotein levels. The novel quantitative strategy to evaluate the relative intensity of the six states of N-glycan type glycosylation on each site provides a new avenue to investigate function of glycoproteins in broad areas, such as cancer biomarker research, pharmaceuticals characterization and anti-glycan vaccine development.

Feasibility of Utilizing Stable-Isotope Dimethyl Labeling in Liquid Chromatography–Tandem Mass Spectrometry-Based Determination for Food Allergens—Case of Kiwifruit

Stable-isotope dimethyl labeling is a highly reactive and cost-effective derivatization procedure that could be utilized in proteomics analysis. In this study, a liquid chromatography– tandem mass spectrometry in multiple reaction monitoring mode (LC-MS-MRM) platform for the quantification of kiwi allergens was first developed using this strategy. Three signature peptides for target allergens Act d 1, Act d 5, and Act d 11 were determined and were derivatized with normal and deuterated formaldehyde as external calibrants and internal standards, respectively. The results showed that sample preparation with the phenol method provided comprehensive protein populations. Recoveries at four different levels ranging from 72.5–109.3% were achieved for the H-labeled signature peptides of Act d 1 (SPA1-H) and Act d 5 (SPA5-H) with precision ranging from 1.86–9.92%. The limit of quantification (LOQ) was set at 8 pg mL−1 for SPA1-H and at 8 ng mL−1 for SPA5-H. The developed procedure was utilized to analyze seven kinds of hand-made kiwi foods containing 0.0175–0.0515 mg g−1 of Act d 1 and 0.0252–0.0556 mg g−1 of Act d 5. This study extended the applicability of stable-isotope dimethyl labeling to the economical and precise determination of food allergens and peptides.