Sample preparation

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Clean, careful, targeted sample preparation is essential for obtaining valuable information from mass spectrometry experiments. A well-designed sample preparation protocol must avoid contamination by keratin, must extract all proteins of interest and should eliminate proteins that are not of interest. The sample preparation laboratory contains all equipment, supplies and expertise necessary for most sample preparation and clean-up techniques used for bacterial, fungal, plant, insect and mammalian samples.

Samples may be prepared by customers or by the Nevada Proteomics Center. The perfect sample for mass spectrometry contains protein but few nucleic acids, salts, complex carbohydrates or lipids. A pellet of precipitated protein or a pellet of washed membranes is usually the ideal sample.

Prefractionation for mass spectrometry analysis

For a very complex sample, prefractionation of the proteins in the sample, followed by LC-MS analysis of each fraction, allows for identification of many more proteins than can be seen without such prefractionation. This is usually performed by separating proteins or protease fragments on the basis of charge. The Nevada Proteomics Center has a number of methods for accomplishing such separation.

Proteins or peptides can be separated by basic reversed phase HPLC on our Thermo Scientific Ulti-Mate 3000 HPLC. Fractions are automatically collected by this instrument and then analyzed.

An alternative method for separating by charge is in-solution isoelectric focusing. The Sample Preparation lab has two different instruments that can accomplish this. We have an Agilent Offgel fractionator capable of separating either intact proteins or protease fragments into as many as 24 fractions based on isoelectric point. After a quick clean-up step, the fractions are ready for analysis. A second method for in-solution isoelectric focusing of proteins is use of our Bio-Rad microRotofor Cell. This method produces 10 fractions which can be analyzed on 1-D gels or by LC/MS. In-solution focusing may provide an excellent separation method for proteins, such as membrane proteins or very large proteins that are difficult to handle by other methods. Both instruments are in our open access sample prep facility.

Enzymatic protein digestion

Before being analyzed by mass spec, most proteins must be broken into smaller fragments. This is accomplished by digestion with a protease, usually trypsin and Lys-C, but may be another proteases of your choice.

Requirements

>100 fmol protein, may be a protein in solution, a gel slice or spot that is visible by coomassie staining.

In the Nevada Proteomics Center, trypsin digestion is performed either by a manual protocol, which is slow but quite effective, or by use of the very rapid Perfinity Flash Digest system.