# A Comprehensive Review of Milk Components: Recent Developments on Extraction and Analysis Methods
## 4 Analytical Methods for Milk Macro-Constituents
### Analysis of Proteins  
Milk proteomics is the comprehensive study of the protein composition, structure and function of proteins in milk. This field uses advanced analytical techniques, primarily mass spectrometry, to identify the wide array of proteins in milk from various sources, such as bovine, human, goat and sheep milk. The general workflow for analyzing milk proteins by MS involves some key steps after milk defatting and protein precipitation and separation, i.e., digestion and peptide clean-up. Protein digestion involves denaturation, reduction and alkylation and enzymatic hydrolysis. Typically, denaturation is done using urea or other chaotropic agents to unfold their structure, while for the reduction of disulfide bonds and for the alkylation of free sulfhydryl groups, dithiothreitol and iodoacetamide are used, respectively. Finally, trypsin is used to digest proteins into peptides. Other enzymes like chymotrypsin or serine protease Glu-C can also be employed for complementary digestion [93]. In some cases, before digestion, a quantification study could be requested, in order to have preliminary information about protein concentration. Different kinds of assays are available, like Bradford and Bicinchoninic assays. The Bradford assay is based on the binding of tryptophan, tyrosine and phenylalanine aminoacidic constituents of proteins to the Coomassieie Brilliant Blue G-250 colorant. A shift from 465 to 595 nm, due to the linkage of the colorant to the aminoacidic components, is observed, which is proportional to the amount of milk proteins. Instead, with the Bicinchoninic (BCA)assay, the quantification is achieved after the absorbance measurement at 560 nm, due to complex formation between Cu ions and cysteine, tryptophan, tyrosine and phenylalanine, enhanced by the addition of BCA [94]. Many other commercial instruments are available for fast protein analysis in milk and are based on absorbance readings in the MID-IR range (9600 nm) [95]. On the other hand, an interesting study by [PERSON] et al. [96] combined the use of differential calorimetry (DSC), thermogravimetric analysis, Fourier transform infrared and scanning electron microscopy (SEM) techniques, to identify casein in cow milk. With FTIR, it is possible to identify and quantify the protein content based on characteristic amide bands, while SEM is primarily used for studying the microstructure of protein aggregates or protein interactions with other milk components. Comparing this work with other work based on LC-MS [97; 98], it is immediately clear that LC-MS analysis is more laborious as more steps for sample preparation are needed. On the other hand, LC-MS technique provides highly sensitive and specific results, as it can identify low-abundance proteins. Moreover, it is suitable for detecting protein adulteration or contamination [99]. Reverse phase LC has been commonly used to perform the separation of casein in different kinds of milk, including breast milk [93] or to profile milk proteins after heat treatment [100]. As mobile phases, ultrapure water acidified with 0.1% formic acid and acetonitrile are mainly used. After MS or MS/MS detection, the identification of peptide ions for protein sequence analysis is accomplished through the matching of the peptide mass spectra with those available in protein databases like Mascot [101], MaxQuant [102] or Proteome Discoverer [103]. On the other hand, with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is possible to quickly identify milk proteins or peptides without preliminary extensive separation. MALDI-MS is characterized by simplified workflow and high sensitivity as it allows for the detection of intact proteins or peptide fingerprints in combination with enzymatic digestion [99]. Recent applications of this technique in the field of milk analysis regard the identification of milk adulteration with non-declared animal sources (e.g., buffalo or goat milk mixed with cow milk).