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Get More From Your Proteomics: Expanded Applications for ProteaseMAX

Gary Kobs and Natalie Larsen

Promega Corporation

Publication Date: 07/2019; tpub_214

Introduction

Identifying proteins resolved by SDS‐PAGE is no simple undertaking. Its success is entirely dependent on an effective in‐gel protein digestion and efficient peptide extraction procedure, two requirements that are tricky in and of themselves to achieve. The pursuit of protein identification is made all the more challenging due to the extensive and time-consuming nature of the procedure.

ProteaseMAX™ Surfactant, Trypsin Enhancer is designed to improve protein identification procedures by minimizing the number of steps needed for in-gel digestion and extraction. Over the course of the in-gel digestion reaction, ProteaseMAX™ Surfactant, Trypsin Enhancer (Cat.# V2071, V2072) degrades proteins and those degradation products are primarily removed during sample handling steps, which eliminates the need to perform a post-digestion degradation. By orchestrating efficient peptide extraction from gel, ProteaseMAX™ Surfactant eliminates the need for a time-consuming acetonitrile extraction step. 

ProteaseMAX is thus able to achieve maximal protein digestion by condensing the time for in‐gel digestion and extraction to as little as one hour, saving the user considerable time and labor (Figure 1). 

time-saving-advantage-of-proteasemax-surfactant-10827ma-w
Figure 1. Time-saving advantage of ProteaseMAXTM Surfactant for in-gel protein digestion.

In a recent study, the ProteaseMAX™ Surfactant was found to induce a 1.5- to 2-fold increase in peptide recovery. As a result, protein sequence coverage was increased by 20–30%, on average, and the number of identified proteins saw a substantial boost (Figure 2; 1).

The researchers additionally observed that the ProteaseMAX Surfactant procedure consistently exhibited superior performance in comparison to conventional procedures, and noted that even after over 100 runs, the surfactant did not affect peptide retention, signal-to-noise ratio or signal intensity (1).

In addition to consistently demonstrating its prowess for in-gel protein digestion, ProteaseMAX™ Surfactant has recently proven to be useful in novel mass spectrometry-related applications as well, which we further describe below. 

benefits-of-proteasemax-surfactant-11253md-w
Figure 2. Benefits of ProteaseMAXTM Surfactant for in-gel digestion.

Top-Down Protein Characterization

On‐tissue spatially‐resolved proteomics are an excellent medium for studying cellular‐level proteomic fluctuations that occur in response to changes in the tissue microenvironment (2). It plays an exceptionally useful role when applied to the study of physiopathological diseases like cancer, as it can more closely examine tumor heterogeneity and cellular cross talk happening in different parts of the tumor.

In a recent study, researchers developed a novel strategy that coupled on‐tissue spatially resolved top‐down proteomics with matrix‐assisted laser desorption/ionization (MALDI) Mass 
Spectrometry (MS) Imaging in order to identify low‐mass proteins and proteoforms, and to localize them (3).

They utilized this strategy to analyze three different regions of the rat brain. They first performed molecular histology using MALDI MS Imaging, followed by spatial segmentation to determine regions of interest (ROIs) within the desired tissue. Protein microextraction, utilizing a 0.01% solution of ProteaseMAX™ surfactant in 50μM DTT, was then performed on these ROIs, closely followed by nanoLC high resolution MS/MS analysis to confirm the efficiency of the microextraction.

This study lead to the successful characterization of 123 proteins between the three brain regions, a stark contrast to a previous study, in which only 36 proteins were identified utilizing whole tissue proteomics and acidified MeOH to perform extraction. These results demonstrate the potential of utilizing this coupled system, in conjunction with ProteaseMAX, to more effectively search for novel proteins, biomarkers and detection of post‐translational modifications.

Protein Identification From Gelatin Samples 

Gelatin is an extremely versatile substance that demonstrates a variety of functional properties, making it a popular, in‐demand ingredient for many products across the food, cosmetic and pharmaceutical industries. Most commercial gelatins are derived from the partially‐hydrolyzed collagen of three main mammalian types: bovine‐hide, porcine‐hide and donkey‐hide.

However, not all gelatin is created equal. A recent study set out to differentiate each of these gelatins, by characterizing the unique marker peptides and modifications of each type. To achieve this, the research team utilized a 0.01% ProteaseMAX surfactant solution to perform individual digestions of each gelatin sample, which were then analyzed using both high‐performance liquid chromatography (HPLC) and high‐resolution mass spectrometry (4).

From this analysis, they were able to determine distinct marker peptides for each of the three gelatin types, and demonstrated the potential of this method as a promising technology to implement in quality control for products, particularly in the food industry, that have gelatin as an added ingredient.

Simultaneous Extraction of DNA and Protein

Proteomic methods, particularly bottom‐up proteomics, have continued to demonstrate their value in improving forensic applications related to protein mass spectrometry. Amino acid sequences not only reveal which human or non‐human proteins are present, they can also demonstrate variability between individuals. For DNA analysis, STR is still the primary tool to identify unique individuals.

In a recent study, a research team set out to determine if it was possible to obtain PCR‐STR results for mass spectrometry protein sequences and DNA analysis, when beginning with a single trypsin digestion sample (5). To test this, they utilized Millipore Microcon® MW100 filter units, replaced proteinase K with trypsin and sodium dodecyl sulfate (SDS) with ProteaseMAX.

Results indicated that trypsin digestion and Microcon® MW100 filtration method can be used to successfully and neatly separate DNA and protein components from fingerprints. In a parallel comparison with the standard proteinase K method, the Microcon® method revealed DNA yields similar to proteinase K extraction method and PCR‐STR results that were better.

The Microcon® method also produced peptides in the flow through that were particle‐free, in aqueous solution and mass spectrometry‐ready, indicating that this method is a suitable approach for forensic investigators to not only process touch evidence, but also test for tissue specific protein markers and protein polymorphisms.

Quantitation of Specific Plasma Membrane Proteins

The ability to reproducibly quantify drug transporter protein expression in tissues is important in order to not only predict transporter‐mediated drug disposition, but also because it is required to construct physiologically‐based pharmacokinetic models.

Most of the current mass spectrometry-based transporter protein quantification methods result in high variability of the estimated transporter quantities. In this study, the goal was to evaluate the various protein sample digestion protocols to quantify drug transporter proteins, and determine an optimized method for quantification of transporters in mouse tissues (6).

Their evaluation of the protein digestion protocols revealed that the most optimal protocol for plasma membrane protein digestion is with Lys‐C and trypsin, in combination with a ProteaseMAX Surfactant enhancer and heat denaturation. This protocol provided them with an easily reproducible method with great precision, with the coefficient of variation for technical replicates under 10%.

References

  1. Saveliev, S. et al. (2013) Mass spectrometry compatible surfactant for optimized in-gel protein digestion. Anal. Chem. 85, 907–14.
  2. Quanico, J. et al. (2013) Development of liquid microjunction extraction strategy for improving protein identification from tissue sections. JProteomics. 79, 200–18.
  3. Delcourt, V. et al. (2018) Spatially-resolved top-down proteomics bridged to MALDI MS imaging reveals the molecular physiome of brain regions. Mole. Cell. Prot. 17, 357–72.
  4. Sha, X.-M. et al. (2018) The identification of three mammalian gelatins by liquid chromatography-high resolution mass spectrometry.  Food. Sci. Tech. 89, 74–86.
  5. Kranes, S. et al. (2017) Simultaneous DNA and protein extraction using trypsin. Forensic Sci. International. 6, e203–04.
  6. Jankovskaja, S. et al. (2019) Optimization of sample preparation for transporter protein quantification in tissues by LC-MS/MS.  J. Pharma, Bio. Ana. 164, 9–15.

ProteaseMAX Surfactant, Trypsin Enhancer is a trademark of Promega Corporation.

Microcon is a registered trademark of Millipore Corporation. 

Optimize Your In-Gel Protein Digestion

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