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Sample preparation and protocols

Sample preparation

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 protease of your choice.

Requirements

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

Protocols

These are basic protocols for either in-gel or in-solution protein digestion. Please contact us if you are looking for more detailed sample preparation methods.

In-gel digestion

The following in-gel digestion protocol is used for up to 3 - 1mm round gel pieces. The methods should be modified if starting with additional gel pieces such that all gel pieces are completely covered in solution for all steps. The amount of protease should also be adjusting accordingly.

Reagents

  • 25mM ammonium Bicarbonate (ABC)
  • 10mM dithiothreitol (DTT) in 25mM ABC
  • 100mM iodoacetamide (IA) in 25mM ABC
  • 20 μg lyophilized modified trypsin (Promega Cat# V5111)
  • 0.3% formic acid (aq)
  • Acetonitrile (ACN)

Methods

  • Add 50μl ABC to gel plugs and incubate at room temperature (RT) for 5 minutes.
  • Discard solution and add 50μl ACN, incubate at RT for 5 minutes.
  • Repeat both steps.
  • Discard solution and add 40μl DTT, heat to 60C and incubate for 10 minutes. Let cool 15 minutes.
  • Discard solution and add 30μl IA. Incubate for 35 minutes at RT.
  • Discard solution and wash two times with ABC and ACN as stated above.
  • Add 15μl of trypsin solution, incubate for 10 minutes.
  • Note: Make trypsin solution immediately prior to addition. Dissolve trypsin in 0.3% formic acid to give a 0.1ug/ul solution and dilute to 5ng/ul in 25mM ABC.
  • Add 10μl ABC and incubate for 4 hours at 37C.
  • Add 15μl 0.3% formic acid.

In-solution digestion

Starting with a sample of <20 μl sample buffer.

Reagents

  • 25mM ammonium Bicarbonate (ABC)
  • 10mM dithiothreitol (DTT) in 25mM ABC
  • 55mM iodoacetamide (IA) in 25mM ABC
  • 20 μg lyophilized modified trypsin (Promega Cat# V5111)
  • 0.3% formic acid (aq)
  • Acetonitrile (ACN)

Methods

  • Add 20μl ACN, incubate at RT for 20 minutes.
  • Add 40μl DTT, heat to 60C and incubate for 10 minutes.
  • Let cool 15 minutes.
  • Add 20μl IA. Incubate for 35 minutes at RT.
  • Add 20μl of trypsin solution, incubate for 10 minutes.
  • Note: Make trypsin solution immediately prior to addition. Dissolve trypsin in 0.3% formic acid to give a 0.1ug/ul solution and dilute to 5ng/ul in 25mM ABC.
  • Add 10μl ABC and incubate for 4 hours at 37C.
  • Add 20μl 0.3% formic acid.