Sample Preparation

Sample Preparation Information for Mass Spec Analysis of Protein Identification

Protein in Gel Slides:

Samples should be prepared by running the desired protein sample down a 1D SDS-PAGE gel or a 2-dimensional  SDS-PAGE gel. Sample purity is critical in obtaining good protein identification. Therefore 2-Dimensional SDS-PAGE is the preferred method of sample preparation.

SDS-PAGE gels should be stained with stains compatible with MS analysis. It includes Coomassie Blue, SYPRO Ruby and Cy dyes. Only the Silver stain that compatible with MALDI analysis is recommended. For strong stains like Coomassie Blue and MS compatible Silver Stain, pooling of 2 spots is sufficient for 1 MS analysis. For weaker stains like SYPRO Ruby and Cy dyes, pooling of 4 spots is recommended. Proteins should be excised from the SDS-PAGE gel and placed in dried 1.5mL of micro tubes.

We offer 2 types of MS analysis: (i) MALDI-ToF/TpF and LC/MS/MS. The MALDI-ToF/ToF is sensitive enough to give spectra on samples that have as little as femtamoles of protein present but more of the sample would secure better results. A general rule of thumb is if the spot can be seen on a Coomassie stained gel, then MALDI results should be able to be obtained. To secure higher successful rate of MS analysis, all low abundant protein(s) that could only be seen with MS Compatible Silver Stain, SYPRO Ruby and Cy dyes will go directly for LC/MS/MS services.

Samples should be submitted with our Service Order Form. The results will be released within 1 to 2 weeks upon receipt of your samples. An email will be generated to you to acknowledge the receipt of your order.

Protein in Solution

The preparation of samples in solution in a form suitable for protein sequencing is more difficult. Please ensure information on sample concentration, volume, and type of buffer is indicated on the order form.

Some points to remember are:

There is no price difference for MS analysis of proteins from gel pieces or in solution.

  1. The solution must contain 1 protein only. Samples containing more than 1 protein will produce more than 1 sequencing signal.
  2. The more steps used in sample preparation, the greater the possibility of sample loss.
  3. Samples should be in a volatile solvent such as water, acetic acid, trifluoroacetic acid, triethylamine, or acetonitrile.
  4. Reagents and solvents should be ultra-pure or analytical grade.
  5. The sample solution should not contain reagents that interfere with the sequencing process. Tris, HEPES, glycine, glycerol, sucrose, Triton X-100, ammonium sulfate and other ammonium salts are some of the reagents that can interfere and should be removed.
  6. Low concentrations of SDS (up to 0.3%) and PBS can be tolerated 

Sample Preparation Information for N-Terminal Protein Sequencing

Protein in PVDF membrane (recommended) :

The easiest and most efficient way of preparing a protein for sequencing is to carry out 1 dimensional SDS-PAGE or 2-D IEF-SDS -PAGE followed by electro-transfer to PVDF. This has the advantage of removing any contaminating salts or detergents from the sample. Please use fresh reagents and prepare the gel at least 24 hours in advance to avoid risk of N-terminal blockage of the sample due to reaction with free acrylamide in the PAGE gel.

Before transferring the protein from gel to PVDF membrane, remove the glycine by washing both the gel and membrane with transfer buffer. It is preferable to use the high binding capacity PVDF membranes such as Millipore Immobilon-Psq, Bio-Rad Sequi-Blot PVDF, or ABI ProBlott. Nitrocellulose cannot be used as it is not compatible with the solvents used in Edman chemistry. We have found that 'tank' blotting gives better results than 'semi-dry' blotting and we recommend its use. The transfer buffer used depends on the protein of interest.

According to Dunn (1997) the following compositions are commonly used:

  • For proteins with pI between 4 and 7: Dissolve 6.06g Tris and 3.09g boric acid in about 600 ml of ddH2O and adjust the pH to 8.5 with NaOH. Make up to 1L.
  • For proteins with pI between pH 6 and 10: Dissolve 2.21 g CAPS in about 600 ml of ddH2O, add 100mls MeOH and adjust pH to 11 with NaOH.

The time of transfer depends on the thickness and percentage acrylamide of the gel and the molecular weight of the protein. Time can vary from 1-6 hours. We have obtained good transfer with CAPS buffer blotting for 2 hours at 500mA.

The PVDF is then stained by a brief (< 5 mins) incubation in Coomassie stain (0.2% Coomassie blue R-250, 45% MeOH, 10% Acetic acid) and destained in 45% MeOH, 10% acetic acid. The membrane is allowed to dry and the protein band can then be cut from the membrane for sequencing. Please do not use Pierce GelCode Blue Stain or other pre-made stains as they may contain detergents, which can interfere with some PTH amino acids.

When a large amount of protein (40-100 pmol) is available, running three to five lanes is helpful. Multiple bands can be cut if needed, especially for higher molecular weight proteins. If the amount of protein is limited (10-20 pmol), try to load more onto the gel or prepare multiple bands of the same sample. It is recommended to send 5 to 10 pmoles.

It is recommended that the entire membrane be sent with a copy marking the band(s) with estimated molecular weight on a photocopy of the gel image.

Protein in Solution:

The preparation of samples in solution in a form suitable for protein sequencing is more difficult. Please ensure information on sample concentration, volume, and type of buffer is indicated on the order form. (Additional charges apply for run gel & PVDF membrane blotting services)

Some points to remember are:

  • The solution must contain 1 protein only. Samples containing more than 1 protein will produce more than 1 sequencing signal.
  • The more steps used in sample preparation, the greater the possibility of sample loss.
  • Samples should be in a volatile solvent such as water, acetic acid, trifluoroacetic acid, triethylamine, or acetonitrile.
  • Reagents and solvents should be ultra-pure or analytical grade.
  • The sample solution should not contain reagents that interfere with the sequencing process. Tris, HEPES, glycine, glycerol, sucrose, Triton X-100, ammonium sulfate and other ammonium salts are some of the reagents that can interfere and should be removed.
  • Low concentrations of SDS (up to 0.3%) and PBS can be tolerated 

Complex samples containing more than one protein in solution cannot be analyzed by this process.