Sample Preparation Tips the Balance for Successful Protein Discovery

New mass spectrometry protocol relies on Acrodisc® MS syringe filters for critical sample preparation

October 21, 2021

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Mass spectrometry (MS) is one of the most powerful techniques available for protein discovery. The technique relies on the identification of short protein sequences by their mass and charge; however, protein abundance often determines signal strength and peptide coverage, making the identification of low abundance protein a challenge.

 

Protein secretion is essential to intercellular communication, governing an amazing variety of processes. Secretome analysis, or the proteomic analysis of secreted proteins, is a relatively new field that has become especially important for the study of disease pathology.

 

Since many secreted bioactive peptides are present at exceedingly low concentrations, they can be difficult to detect in a complex mixture. This makes MS sample preparation a critical step for the discovery of novel secreted proteins. A recently published protocol designed for quantitative mass spectrometry describes how sample preparation can help scientists not just identify proteins but compare relative abundance in different protein samples. [1]

 

The benefits of sample filtration for confident MS data

 

Sample filtration is often dismissed as a “standard” step in the MS sample preparation workflow but neglecting this step can cause significant problems downstream. This is particularly true for quantitative MS, where scientists are seeking to reveal sometimes subtle differences in protein abundance. The scientists who devised this new protocol aimed to increase the level of confidence that they were identifying legitimate secreted proteins versus contaminating intercellular proteins from dead cell debris. They did this by performing MS analysis of both the secretome and proteome of the same cell culture replicates and comparing the relative abundance of identified proteins. Cell culture conditions and sample preparation were optimized to avoid false-positive identification of secreted proteins.

 

The quantitative MS protocol was performed using A549 human lung cells and is composed of the following steps:

 

  • Cell culture:  Confluent cultured cells are washed and then incubated in serum-free media to minimize contaminants from serum in the MS sample.

  • Conditioned media and cell harvest: After 24 hours in serum-free media, the supernatant (conditioned media containing secreted proteins) and cells are harvested separately.

  • Sample preparation: Conditioned media and cell samples are prepared in tandem. Protein concentrations for the two sample sets were equalized prior to gel analysis.
 

1.) Conditioned media (cell secretome) is harvested, centrifuged, and filtered using an Acrodisc MS syringe filter to remove residual dead cells and cell debris. Proteins are extracted and prepared for protein concentration measurement and in-gel tryptic digest.

 

2.) Cell pellets are harvested, pelleted, and resuspended in lysis buffer to extract proteins. Cellular extracts (cell proteome) are prepared for protein concentration measurement and in-gel tryptic digest.

 

  • MS analysis: Extracted tryptic peptides from individual gel bands are analyzed by LC-MS/MS.

  • Database search and analysis: MS raw files for both secretome and proteome analysis sets are exported and subjected to data analysis programs for protein identification and functional annotations.

 

The choice of filter used for MS sample preparation depends on parameters such as sample size, protein and solvent characteristics, and the intended downstream analysis technique.

 

Acrodisc MS syringe filters are designed to minimize the interference with ionization results through the use of manufacturing materials validated to contain extremely low levels of extractables.

 

Acrodisc filters contain WWPTFE (Water Wettable PolyTetraFluoroEthylene) membranes that are low-protein binding and can be used with both aqueous solutions and organic solvents. Acrodisc MS syringe filters are available in 2 sizes: the 13 mm format can effectively process a typical sample volume of ≤ 10 mL, while the 25 mm format is ideal for sample volumes ≤ 150mL.

 

Following MS analysis and functional annotation, the researchers examined extra-cellular to intracellular protein ratios, to determine which of the identified proteins were enriched in the secretome data set as compared to the proteome data set. These results are considered by the authors to represent legitimate secreted proteins with a high level of confidence.

 

Experimental approaches used to analyze protein secretion are constantly being refined and optimized. Interest in cell secretome analysis is rapidly expanding as investigational techniques become more advanced and therapeutic applications more feasible.

 

At Pall, we are proud to support this type of state-of-the-art biomedical research with a wide range of analytical sample filtration solutions. Please visit our website to learn more.

 

Reference

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