A Cleavable Surfactant Capable of Disrupting Cell Membranes and Solubilizing Proteins with No Interference
PPS Silent® Surfactant Use a single tube, zero clean up protocol to extract, solubilize, and digest proteins from small cell populations. Now you can use mass spectrometry to analyze proteins extracted from Laser Capture Microdissection (LCM) samples and other small cell populations without pooling cells from multiple sections. PPS Silent® Surfactant disrupts cell membranes, extracts and solubilizes proteins, then breaks down into nonsurfactant hydrolytic cleavage products. The single tube protocol prepares samples for nanoLC-MS / MS analysis without the use of salts or detergents which would necessitate yield reducing sample clean up prior to injection.
Reduces detergent interference in mass spectrometry
Disrupts cell membranes
Solubilizes hydrophobic proteins
Improves enzymatic efficiency
Improves MS analysis of complex protein mixtures
Improves membrane protein ID
Hydrolyzes into soluble, nonsurfactant cleavage products.
Increase shotgun proteomics sequence coverage
Enable membrane proteomic analysis of cells captured by LCM
Potentiate trypsin digestion of membrane proteins
Enable iTRAQ quantitation of insoluble proteins.
How it Works PPS Silent® Surfactant is acid cleavable so it can be removed by hydrolysis. The intact molecule is zwitterionic, so PPS Silent® Surfactant can also be removed by strong cation exchange HPLC (SCX HPLC).
Hydrolysis of PPS Silent® Surfactant clears detergent interference. Acid hydrolysis of PPS Silent® Surfactant clears detergent interference, enabling mass spectrometry (MS) analysis of peptides resulting from trypsin digestion of complex protein mixtures. Hydrolysis products are nonsurfactant. Loss of surfactant property upon hydrolytic cleavage. Cleavage occurs within 30 minutes in 0.25M HCl.
Does PPS Silent Surfactant affect mass spectrometry?
PPS is specifically designed to have minimal negative impact on mass spectrometry. Reducing the pH of the digest buffer cleaves this reagent, allowing MALDI-TOF MS, HPLC/MS, or HPLC/UV analysis without detergent interference.
What is the CMC of PPS Silent Surfactant?
The CMC is 0.128 mM.
The CMC is the critical micelle concentration. This is the concentration above which the addition of more detergent will result in the formation of micelles in solution. It is generally regarded that for optimum solubilization, the detergent should be used at or above this concentration. For optimal removal of detergent from the sample, or reduction in the interference with MS, the concentration of uncleaved PPS should be below this value.
Is PPS Silent Surfactant hygroscopic?
Yes. For best results, follow the product insert Use and Storage Instructions.
Is PPS Silent Surfactant ionic, non-ionic, or zwitterionic?
PPS Silent Surfactant is zwitterionic.
Does PPS Silent Surfactant dissolve in ordinary buffers (e.g. Tris or Ammonium Bicarbonate) and organic solvents?
PPS Silent Surfactant is water soluble and will replace most mass spectrometry incompatible detergents in a variety of biological buffers and solvent systems.
Can PPS Silent Surfactant be used in place of SDS?
PPS Silent Surfactant is zwitterionic, so it cannot be used as a substitute for SDS in gels. However, it can be used in isoelectric focusing. The addition of SDS to focused proteins does not interfere with conventional 2D PAGE analysis.
What pH is required for acid hydrolysis of PPS Silent Surfactant?
At neutral pH, the surfactant is stable for many hours; at pH <4 cleavage becomes facile. The lower the pH, the faster cleavage will occur.
See technical note at the end of this section.
What acids are used to cleave PPS?
Our recommended protocol uses HCl. However, if this condition is not suitable for your application, organic acid such as TFA, formic acid, and acetic acid may be used. The time required for cleavage must be adjusted accordingly (see previous).
Is PPS Silent Surfactant trypsin-compatible?
Yes. Trypsin is active in the presence of PPS.
How much protein can you use per reaction?
Up to 10 mg/ml of protein is effectively solubilized by 0.1% PPS Silent Surfactant.
Is it OK to use protease inhibitors in combination with PPS Silent Surfactant?
In our experience, the addition of protease inhibitors does not caused any problems. For best results, use Proteoloc™ Protease Inhibitor Cocktails.
Is it OK to use urea in combination with PPS Silent Surfactant?
The addition of urea to a detergent solution may inhibit micelle formation and interaction of the detergent with the protein. In most instances, the recommended PPS Silent Surfactant concentration of 0.1% will be effective, even in the presence of urea. If more detergent activity is required, the concentration of PPS Silent Surfactant may be increased to as high as 2%.
Is it OK to use PPS Silent Surfactant at high temperature?
Yes. Heat may accelerate hydrolytic cleavage of PPS Silent Surfactant in acid conditions.
Does PPS Silent Surfactant work for the extraction of organellar proteins?
Yes. Our customers have successfully used PPS Silent Surfactant to analyze mitochondrial proteins.
Is the use of PPS Silent Surfactant compatible with reverse-phase chromatography?
Does PPS Silent Surfactant stick to reverse phase column material?
The hydrophobic portion of cleaved PPS Silent Surfactant is 1-hexanol, which does not associate strongly with any kind of reverse phase material. Unexpected downstream effects may be caused by the presence of uncleaved surfactant. In this case, cleavage conditions should be optimized by reducing the pH of the digest buffer by one unit or by extending the reaction time interval ten-fold.
Is PPS Silent Surfactant compatible with MudPIT?
Can PPS Silent Surfactant be used in gels?
PPS Silent Surfactant is zwitterionic and therefore cannot be used as a direct replacement for SDS in gels. Proteins solubilized with PPS Silent Surfactant can be run in 2D gels and westerns.
Is PPS Silent Surfactant compatible with CE?
Yes. Capillary electrophoresis separations may be sensitive to the presence of uncleaved PPS Silent Surfactant. If the end application is CE, good results may require optimizing PPS Silent Surfactant cleavage conditions by reducing the pH of the cleavage reaction solution, or by extending the cleavage reaction time interval.
Can PPS Silent Surfactant be stored after it has been re-suspended?
PPS can be stored if resuspended in an aprotic solvent such as DMSO. Our recommendation would be to make a stock solution of 20x PPS in DMSO, aliquot and freeze for use later. It is important to be sure that the DMSO used in dry. If contaminated with water, the PPS will hydrolyze even in the DMSO stock. Once resuspended in protic solvent (e.g. water or buffer) the PPS should be used within the day.
For all safety related questions, refer the customer to the information contained within the product MSDS. This document is included at the end of the section.
PPS Silent® Surfactant is a cleavable surfactant capable of disrupting cell membranes and solubilizing proteins. At low pH, PPS hydrolyzes rapidly into cleavage products with no surfactant properties and no detergent interferences. Unlike first-generation cleavable surfactants for MS, there is no oily film or sticky pellet to complicate sample preparation.