Comparative two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE) of human milk to identify dysregulated proteins in breast cancer

Breast cancer (BC) remains a major cause of mortality, and early detection is considered important for reducing BC‐associated deaths. Early detection of BC is challenging in young women, due to the limitations of mammography on the dense breast tissue of young women. We recently reported results of a pilot proteomics study, using one‐dimensional polyacrylamide gel electrophoresis (1D‐PAGE) and mass spectrometry (MS) to investigate differences in milk proteins from women with and without BC. Here, we applied two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE) and MS to compare the protein pattern in milk from the breasts of a single woman who was diagnosed with BC in one breast 24 months after donating her milk. Statistically different gel spots were picked for protein digestion followed by nanoliquid chromatography tandem MS (nanoLC‐MS/MS) analysis. The upregulated proteins in BC versus control are alpha‐amylase, gelsolin isoform a precursor, alpha‐2‐glycoprotein 1 zinc isoform CRA_b partial, apoptosis‐inducing factor 2 and vitronectin. Several proteins were downregulated in the milk of the breast later diagnosed with cancer as compared to the milk from the healthy breast, including different isoforms of albumin, cholesterol esterase, different isoforms of lactoferrin, different proteins from the casein family and different isoforms of lysozyme. Results warrant further studies to determine the usefulness of these milk proteins for assessing risk and detecting occult disease. MS data is available via ProteomeXchange with identifier PXD009860.     

Migration of perfluorinated compounds from paperbag to Tenax® and lyophilised milk at different temperatures

ABSTRACT

Migration tests of perfluoroalkyl substances (PFASs) from a grease-proof paperbag used for packaging of pet food have been carried out. No migration of perfluorocaboxylic acids (PFCAs), from PFBA to PFTeDA, to the simulant Tenax® was found after 10 days at 40°C. However, the increase of temperature in the range 80–160°C gave rise to the migration of the PFCAs. Finally, the migration to real foods such as lyophilised whole and low-fat milk samples at 80 and 120°C was studied. The results indicate that the migration percentages of the PFCASs into food samples are much higher than those obtained into Tenax®.     

Presence of pesticides in breast milk and infants’ formulae in Himachal Pradesh,India

This study documents the levels of pesticide residues in milk samples of mothers from Himachal Pradesh, India, and time trend comparison of pesticide load based on various studies conducted around the world. The regional difference in xenobiotic levels of breast milk varied with demographic characteristics of mothers and altitudinal variations. The single or multiple pesticides contamination of p,p′-DDE, p,p′-DDT and chlorpyrifos was revealed in 27.45% mothers’ milk samples. Among these p,p′-DDE was the major contaminant found in 26.79% samples followed by p,p′-DDT (1.31%) and chlorpyrifos (0.65%). However, residues of other 26 pesticides comprising organochlorines, organophosphorus and synthetic pyrethroids included in this study were below detectable limit (BDL). The determination of a low DDT/DDE ratio (0.050) indicated past exposure of mothers to DDT from the environment. The pesticide residues level in samples drawn from 14 branded infant formulae was BDL. The calculated infants’ daily intake (DI) of DDT was 0.0015 mg kg^?1 body weight per day compared with a decade-old study (0.021 mg kg^?1 body weight per day) suggesting a sharp decline in the residue levels of these pesticides in the Himalayan region. The trend comparison with past studies conducted around the world indicate a decline in the levels of organochlorine pesticide residues in mothers’ milk and further drop of DI in infants. However, such comparisons confer very limited utilisation of data generated on pesticide load in mothers’ milk and simultaneous infants’ DI due to lack of proper research protocol.     

Identification of phosphoproteins and determination of phosphorylation sites by zirconium dioxide enrichment and SELDI-MS/MS

Reversible protein phosphorylation mediated by protein kinases and phosphatases is the most studied post-translational modification. Efficient characterization of phosphoproteomes is hampered by (1) low stoechiometry, (2) the dynamic nature of the phosphorylation process and (3) the difficulties of mass spectrometry to identify phosphoproteins from complex mixtures and to determine their sites of phosphorylation. Combination of the phosphopeptide enrichment method with MALDI-TOFMS, or alternatively, with HPLC-ESI-MS/MS and MS(3) analysis was shown to be a step forward for the successful application of MS in the study of protein phosphorylation. In our study we used phosphopeptide enrichment performed in a simple single-tube experiment using zirconium dioxide (ZrO(2)). A simple protein mixture containing precipitated bovine milk caseins was enzymatically digested and the mixture of tryptic fragments was analysed before and after enrichment using nanoflow HPLC-ESI-MS/MS and surface-enhanced laser desorption/ionization (SELDI)-MS/MS on QqTOF instruments to compare the efficiency of the two methods in the determination of phosphorylation sites. Both approaches confirm the high selectivity obtained by the use of batch-wise, ZrO(2)-based protocol using di-ammonium phosphate as the eluting buffer. More phosphorylation sites (five for beta-casein and three for alpha(S1)-casein) were characterized by SELDI-MS/MS than by nanoflow HPLC-ESI-MS/MS. Therefore, ZrO(2)-based phosphopeptide enrichment combined with SELDI-MS/MS is an attractive alternative to previously reported approaches for the study of protein phosphorylation in mixtures of low complexity with the advance of fast in situ peptide purification. The method was limited to successful analysis of high-abundance proteins. Only one phosphorylation site was determined for the minor casein component alpha(S2)-casein by ESI-MS/MS and none for kappa-casein. Therefore an improvement in enrichment efficiency, especially for successful phosphoproteomic applications, is needed.     

Characterization of the protein profile of donkey's milk whey fraction

Characterization of the protein profile of the whey fraction from a milk sample taken from an individual donkey belonging to the 'Ragusana' species of the East of Sicily is reported. Direct RP-HPLC/electrospray ionization (ESI)-MS analysis of the whey fraction allowed the detection of some unknown components, together with the identification of already known whey proteins. Matrix-assisted laser desorption/ionization (MALDI)-TOF/MS and RP-HPLC/ESI-MS/MS analysis of the enzymatic digests of the unknown components resulted the identification and characterization of (1) two beta-casein fragments; (2) the sequence of donkey's serum albumin; and (3) the oxidized methionine forms of lysozyme B and alpha-lactoalbumin. One of the two beta-casein fragments corresponds to the sequence Val(176)-Arg(189) of the horse's beta-casein. The second one corresponds the C-terminal sequence Tyr(199)-Val(226) of the horse's beta-casein, with four amino acid substitutions (Q --> R(203), L/I --> P(206), F --> L(210) and P --> A(219)). Both fragments, reasonably arising by endogenous proteases cleavage of the donkey's beta-casein, could be potential biologically active peptides. Direct mass spectrometric sequence characterization of the detected donkey's serum albumin reveals the presence of the amino acid substitution Val --> Ile at position 497 with respect to the cDNA deduced sequence. The oxidized forms of lysozyme B and alpha-lactoalbumin are selectively oxidized at methionine 79 and methionine 90, respectively.     

盐酸煮沸提取原子吸收法测定乳和乳制品中矿质元素

本研究了盐酸煮沸提取乳和乳制品中的Ｋ、Ｎａ、Ｃａ、Ｍｇ、Ｆｅ和Ｚｎ六个矿质元素的实验条件,１ｍｏｌ·Ｌ＾－１盐酸提取液用火焰原子吸收法（ＦＡＡＳ）测定,得到满意的结果。方法具有操作简便,安全,快速的特点。与高温干灰化法对十种样品进行结果对照,无论液体样品或样品,其结果都非常一致,不存在显差异,结果具有很好的准确度和精密度。     

β修正光度法测定食品中的微量锰

在pH=8．6的硼砂-磷酸二氢钾缓冲溶液中,锰(Ⅱ)-铬黑T-吐温80显色体系的最大吸收波长为550nm,利用此显色体系,用卢修正光度法测定食品中的微量锰,Mn(Ⅱ)含量在0．45～35μg／(25mL)内服从比耳定律,线性方程为A。=0．0281X-0．0026,相关系数r=0．9997,用该方法测定调味料八角和奶粉中的微量锰。测定结果的相对标准偏差为0．52％～2．86％,回收率为96．21％～102．2％。     

HPLC-MS/MS法测定婴幼儿配方乳粉中胆碱、左旋肉碱、牛磺酸与肌醇

建立了一种同时测定婴幼儿配方乳粉中4种可选择成分(胆碱、左旋肉碱、牛磺酸和肌醇)的高效液相色谱-串联三重四极杆质谱(HPLC-MS/MS)分析方法。样品经温水溶解后用亚铁氰化钾和乙酸锌沉淀蛋白,上清液过滤后采用HSS T3色谱柱分离,三重四极杆质谱仪检测,胆碱和左旋肉碱使用内标法定量,牛磺酸和肌醇使用外标法定量。在最优化条件下,胆碱和左旋肉碱在0.01~2.0 mg/L范围内,牛磺酸和肌醇在0.1~2.0 mg/L范围内呈良好的线性关系,相关系数均大于0.997;胆碱和左旋肉碱的检出限均为1.5 mg/kg,牛磺酸和肌醇的检出限均为15 mg/kg。4种化合物的回收率为87.5%~102.4%,相对标准偏差(RSD,n=6)为3.0%~7.3%。该方法灵敏度高、净化效果好、定量准确,适用于婴幼儿配方乳粉中胆碱、左旋肉碱、牛磺酸和肌醇的同时快速检测。     

Programmable Temperature Vaporizer (PTV) Applied to the Triglyceride Analysis of Milk Fat

The PTV (Programmable Temperature Vaporizer) is a split/splitless injector which allows the sample to be introduced at a relatively low temperature, thus affording accurate and reproducible sampling. After injection the PTV is rapidly heated to transfer the vaporized components into the capillary column. This type of injector has proved to be an efficient tool for the evaluation of fatty acids, essential oils, and pesticides in food analysis. In this work the suitability of PTV for the analysis of milk fat purity by the Official EU method was evaluated. This method is based on the gas chromatographic determination of triglycerides only according to their total number of carbon atoms followed by the application of formulae deriving from multiple linear regressions. The analysis is currently carried out with a packed column or a short capillary column and an on-column injection system. Several samples of pure milk fat and mixtures of milk fat with foreign fat were analyzed with the same capillary column and by using both PTV and on-column injection systems. The results show that the gas chromatographic profile obtained by PTV is comparable with that obtained by the on-column injector, while repeatability and reproducibility data meet the requirements indicated in the Official Method. Therefore, this study demonstrates that it is possible to use the PTV instead of the on-column injector to determine the purity of milk fat with this method.     

Study on the Performance of Three Different Capillary Gas Chromatographic Analyses in the Evaluation of Milk Fat Purity

The adulteration of milk fat with foreign fat has been and still is a major concern in the dairy industry. Milk fat purity is currently evaluated by triglyceride analysis by using the Official EU method. The detection limit of the various vegetable and animal fats ranges between 4 and 6%. This research was carried out to verify whether it is possible to decrease the detection limits of beef tallow, which is the most widely used adulterating animal fat. For this purpose, determinations of diglycerides and 3,5-cholestadiene, together with the Official EU method, were applied both to several samples of pure milk fat and to mixtures of milk fat with different percentages of beef tallow. The best results were obtained combining the data deriving from the three determinations by multivariate statistical techniques; in particular, the statistical model obtained by the UNEQ technique seems to be able to decrease the detection limit of beef tallow from 5.2 to 2%. The diglyceride and 3,5-cholestadiene evaluation, combined with the Official EU method for triglycerides, can be usefully applied both to detect small additions of beef tallow and to demonstrate the adulteration of milk fat samples showing results close to the detection limit of the official method.