Sample preparation guidelines

Reduction & Alkylation
Controls
Amounts
Primary amine
E.coli Standard
Detergents

Reduction & Alkylation.
A protein sample is typically reduced & alkylated to break disulfide bridges and 'cap' the reduced cysteines. For gel-bands, omission of this step can lead to loss of cysteine-containing peptides because free cysteines react covalently with acrylamide in the gel and can therefore not be extracted.  However, reduction & alkylation requires additional steps and can increase the chance of contamination by keratins and other compounds. For identification-only purposes we recommend to omit reduction & alkylation UNLESS identification of cysteine-containing peptides is needed.

Workflow without reduction & alkylation Workflow with reduction & alkylation
Destaining

Dehydration

Addition of trypsin

Removal of excess trypsin

Overnight digestion

Quench digestion
Destaining

Dehydration

Reduction

Removal of excess reduction agent

Alkylation

Removal of excess alkylation agent

Washing

Dehydration

Addition of trypsin

Removal of excess trypsin

Overnight digestion

Quench digestion
Workflow for the in-gel digestion with and without reduction and alkylation.

In the report by Anna and Andrej Shevchenko it is shown that for in-gel digestion, the main difference between conducting reductions & alkylation versus omitting this step is the loss of cysteine-containing peptides: http://www.ncbi.nlm.nih.gov/pubmed/11554724



Controls: To come....

Amount: To come....

Detergents:....http://www.ncbi.nlm.nih.gov/pubmed/21640699. See also this link: http://www.proteinsandproteomics.org/content/free/tables_1/table14.pdf


Primary amines: Various chemistries used to label peptides and proteins are amine-based. Examples are iTRAQ, TMT and dimethyl labeling techniques. Those tags label the primary amines of the peptides/proteins typically at N-termini and the epsilon amino group of Lys. In order for this chemistry to be successful the chemicals used in the sample preparation have to be free of other sources of primary amines (amoniumbicarbonate or TRIS (tris(hydroxymethyl)aminomethane)) that can interfere with labeling reaction. An alternative to amoniumbicarbonate is TEAB (triethylammoniumbicarbonate) whereas HEPES (hydroxyethyl piperazineethanesulfonic acid) can be an alternative to TRIS.


PEG. To come......


Salts.To come......


Volumes.To come......



Amount recommendations:  Estimating protein amount can be challenging. For example, some sample matrices can disturb concentration measurements using Bradford assays. Furthermore, estimating protein amount using staining intensities can be difficult, especially when based on silver staining where the intensity is time dependent. The Proteomics Resource Center reccomends Colloidal Blue staining (protocol: http://tools.invitrogen.com/content/sfs/manuals/colloidalstain_man.pdf, page 8-9 for Bis-Tris gels) because it delivers maximum staining after 3h, minimizing staining-time bias. Colloidal Blue is less sensitive then silver staining, but more sensitive than Commasie Brilliant Blue

To obtain an assessment of protein amount the Proteomics Resource Center offers a standard sample of 20 ug in 20 uL of a commercial E.coli lysate to investigators. This standard is available upon request and can be helpful in the assessment of protein amount and for the general troubleshooting.

silver vs collodial
Figure 1. Bovine Serum Albumin and standard E.coli lysate loaded on SDS-PAGE in different amounts and stained by Colloidal Blue or silver. Including a standard is simply meant to help access amounts. It is highly recommended to use protein concentration measurements if possible as well.

Suggested use of E.coli Standard

1. Prepare 20 ug E.coli lysate (in 20 uL) such that the total volume, including loading buffer and reducing agents, is 60uL. Follow standard protocol for sample preparation. NOTE: The above volume/concentration is chosen so that it is possible to confidently load 1ug (3uL) in one lane and also be able to load 10 ug (30uL) without overloading the well.

3. Load 1 ug of the standard lysate onto one lane and 10 ug onto the adjacent lane.

4. Load samples that are to be compared to the E.coli standard onto adjacent lanes.

5. Run gel.

For some proteomics experiments a rough estimation of sample amount can be sufficient. However, many applications require assessment of absolute or/and relative sample amounts. Examples are chemical labeling experiments where too much or too little of the labeling reagents can offset the results. Furthermore, during analysis of complex samples overloading of the equipment can lead to sub-optimal chromatography/separation resulting in too few protein identifications.