LysoPure™ Protein Extraction-PTS Solution

Proteomics is a comprehensive analytical method used to identify and characterize all proteins in a sample. The process involves extracting proteins from the sample, digesting them into peptides using proteolytic enzymes, and analyzing the structural information of the resulting peptides using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Over the years, numerous improvements have been made to LC-MS/MS, such as improved columns and packing materials, as well as enhanced sensitivity and speed of mass spectrometers. As a result, multiple samples can now be analyzed simultaneously, and both qualitative and quantitative assessment of low-abundance proteins can now be carried out.

Protein extraction and digestion, the preparatory steps for LC-MS/MS, play a crucial role in determining proteomics outcomes. The main challenge is to maximize protein recovery while ensuring efficient digestion with minimal loss. Membrane proteins, in particular, are notoriously difficult to extract due to their high hydrophobicity. Membrane protein extraction in proteomics often necessitates the use of powerful solubilizing agents. However, such agents can inhibit the activity of proteolytic enzymes in subsequent steps and negatively affect peptide separation and ion detection in LC-MS/MS.

To overcome these challenges, a phase transfer surfactant (PTS) based on sodium deoxycholate was developed as an ideal solubilizing agent¹⁾. This surfactant efficiently solubilizes otherwise intractable proteins without inhibiting proteolytic enzyme activity and can be easily removed afterwards. The sample preparation method using this solubilizing agent is known as the PTS method, which has been widely reported as an efficient approach for preparing hydrophobic proteins, including membrane proteins, for proteomics analysis.

1. 

   

2. 

   Extraction of proteins
   from the sample

3. 

   Enzymatic digestion

4. 

   Acidification of the solution,
   followed by stirring and
   centrifugation

5. 

   Removal of
   the upper phase

6. LC-MS/MS

Figure 1. Protein extraction via the PTS method Adapted from Reference 1).

LysoPure™ Protein Extraction-PTS Solution is a solubilizing agent designed for protein extraction, an essential pretreatment step in proteomics. Developed based on the PTS method, this product effectively solubilizes difficult-to-extract proteins while preserving proteolytic enzyme activity and enables easy removal of the agent via liquid-liquid partitioning. This product is an improved version of previously reported PTS solutions^1-2), offering improved protein solubilization and greater proteolytic enzyme activity than conventional PTS solutions. Additionally, it reduces cleavage errors by proteolytic enzymes. This product can also be used in proteomic studies of extracellular vesicles, including exosomes.

Data

Protein Solubilization

Proteins were extracted from human liver microsomal fractions using various PTS solutions with different surfactant compositions. The resulting protein concentrations were compared across the different solutions.

[Result]

This product demonstrated superior solubilization compared to conventional PTS solutions containing other surfactants.

Proteolytic Enzyme Activity and Cleavage Error Rate

Proteolytic Enzyme Activity

PTS Solution	Trypsin			Lysyl Endopeptidase (Lys-C)
	Concentration of PTS solution	Activity	Initial velocity	Concentration of PTS solution	Activity	Initial velocity
SDC＋SLS	4 mM	12.6	4.4	4 mM	2.5	1.9
SDC＋SC	8 mM	5.7	2.3	8 mM	2.6	2.4
SDC＋CDC	8 mM	7.5	4.4	8 mM	2.4	1.8
This Product	8 mM	10.3	4.6	8 mM	2.6	1.8

Number of Cleavage Error

PTS Solution	Number of Cleavage Error
UDC	586
SDC＋SLS	723
SLS＋SC	273
This Product	291

CDC

Kenodeoxycholic acid

SC

Sodium cholate

SDC

Sodium deoxycholate

SLS

Sodium lauroyl sarcosinate

UDC

Ursodeoxycholic acid

[Result]

SDC+SLS exhibited the highest trypsin activity but also resulted in the highest number of cleavage errors. In contrast, although this product had lower trypsin activity than SDC+SLS, it exhibited the highest lysyl endopeptidase activity with fewer cleavage errors. These findings show that this product offers well-balanced performance, combining high enzymatic activity while reducing the number of cleavage errors, making it an ideal PTS solution.

1. Masuda, T., Tomita, M. & Ishihama, Y.: J. Proteome Res., 7(2), 731(2008).
   Phase transfer surfactant-aided trypsin digestion for membrane proteome analysis
2. Masuda, T. et al.: Mol. Cell. Proteomics, 8(12), 2770(2009).
   Unbiased Quantitation of Escherichia coli Membrane Proteome Using Phase Transfer Surfactants