Liquid Chromatography and Mass Spectrometry

After the electrophoresis, liquid chromatography has been extensively used to separate and quantify milk proteins. The LC-MS systems are made of three main parts: the liquid chromatography system, the interface, and the mass spectrometer. The former separate proteins in complex mixtures; the interface transforms the liquid flow from LC system to a volatile media suitable for the mass spectrometer. Finally, the mass spectrometer gives information about the molecular mass of a protein. Two type of ionization techniques are used—matrix-assisted laser desorption ionization (MALDI) and electro spray ionization ESI. The former is a soft ionization technique able to produce a single charged peptide, but it is not suitable for a continuous ionization; therefore, it cannot be used as an interface with an LC system. Generally, MALDI is combined to a time of fly mass spectrometer. MALDI-TOF is the best choice in the analysis of triptic digest of a protein previously purified, thanks to the high mass accuracy and the wide mass range of this technique. The universally used interface in the LC-MS system is the ESI, especially in the nano-ESI configuration, it produces multicharged fragment in a continuous mode. Ion trap, triple quadruple, time of fly, and Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers could be used in proteomic analysis in combination with a LC system and ESI interface. Hybrid mass spectrometers have recently been developed and progressively used in proteomics which are combinations of two of the previously mentioned technology. Examples of hybrid systems are Q-TOF, Q-trap and Orbitrap. These systems are promising machines and offer interesting results in proteomics analysis. The most commonly used combination in milk proteins analysis are: 2-DGE/MALDI-TOF and reversed phase high performance liquid chromatography electrospray ion trap mass spectrometry (RP-HPLC/ESI-IT-MS). All the proteins of milk are separated and analyzed by RP-HPLC coupled to ESI-MS by Leonil et al. (1995). A liquid chromatography-mass spectrometry method to detect the fraudulent addition of cow's milk to water buffalo milk and mozzarella is described by Czerwenka, Muller, and Lindner (2010). These authors used the p-lactoglobulin variant A, absent in water buffalo milk, as a molecular marker. The same result was achieved by Enne et al. (2005) using RP-HPLC and UV detection. Guarino et al. (2010) aimed to evaluate the authenticity of goat and sheep milk by RP-HPLC/ESI-MS. In the study, caseins were extracted from cheeses, solubilized, digested with plasmin, and analyzed by LC/ESI-MS/MS. Target peptides produced by plasmin hydrolysis were selected as molecular markers. Calvano, Monopoli, Loizzo, Faccia, and Zambonin (2013) investigated milk adulteration by powdered milk addition. The authors found that some diagnostic peptides attributed to modified whey proteins and/or caseins. Then, a faster procedure was optimized, consisting of the separation of caseins from milk whey and the subsequent in-solution digestion of the two fractions. Finally, analyses were carried out on milk samples adulterated with powdered milk at different percentages, and diagnostic peptides were detected down to 1% of adulteration level.

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