The complete genome sequence of cattle is available and can be used for mass spe

Question

The complete genome sequence of cattle is available and can be used for mass spectrometry-based proteomics analysis. You decide to use a tandem mass spectrometry approach to examine gene expression in udder tissue during lactation. (1). Describe an experimental approach for identifying proteins that are expressed during lactation, how the resulting data are interpreted, and how you related the genomic data and MS data. (2). You next want to compare protein expression in udder tissues during the stages of lactation and pregnancy. Describe an experimental approach for protein identification and quantitation and a strategy for comparing the resulting protein expression profile for pregnancy versus that for lactation.

Explanation / Answer

Proteins are the major components of the milk. However during lactation period total protein content of the milk is reduced. For identifying the proteins expressed during lactation, cells and fat were removed from milk using centrifugation technique in which the cells form pellet when milk was centrifuged at high speed. Proteins were then extracted from milk using chloroform-methanol method. Extracted protein was then quantified using Bradford method of protein estimation.

Following quantification, particular amount of protein is loaded on Sodium Dodecyl Sulphate Polyacrylamide gel electrophoresis (SDS-PAGE) for separation of proteins in two dimentio (2D) present in milk during lactation under the influence of electric current. 2D gel electrophoresis involves separation of proteins based on ther mass and isoelectric point. Proteins having were separted according to their isoelectric point first and then according to their mass, proteins having similar isoelectric point but different mass, migrate at different speed thereby the heaviest protein will migrate shorter distance as compared to the lighter protein thereby separating the proteins. Once proteins were separated were detected using silver staining procedure. Silver staining procedure is highly senstive method which could detect ng (nano gram) levels of proteins expressed. The stained proteins were subjected to silver destaining procedure and then prepared for In-gel trypsin digestion. Proteins were first treated with Dithiothreitol to convert the disulphide bonds into free sulphydryl groups and then alkylating twith Iodoacetamide so that free sulphydryl groups are not reoxidised to form dulphide bonds again, thereby maximising the trysin access to the proteins for cleavage. Trypsin is an enzyme that digests proteins at the carboxyl end of arginine and lysine.Proteins digested with trysin forms peptides which are separated and identified using mass spectrometry according to their mass and spectra is obtained. The obtained spectra is then matched with spectra present in the database using algorithms (eg: Protein Prophet). Proteins were identified with 99% probability if they have atleast 2 identified peptides.

Correlating the MS data with genomic data can identify genes with the expressed proteome across the udder tissue. Incorrect peptides identified can be correlated and corrected using genomic data. Peptides are considered if there is another peptide in the same reading frame. Genomic data can be utilised to specifically locate the exact postion of the expressed protein.

(2) For comparation, proteins were extracted during different stages of lactation and pregnancy . For example we take at 5 different stages of lactation then 5 different samples are present which will be subjected to similar experimental procedure so as to compare and identify different proteins expressed during different stages.

Experimental procedure is as follows:

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