High-performance Syringe Filters Help Identify Low-abundance Secreted Proteins

In a recent publication by scientists at the Molecular Proteomics Laboratory in Dusseldorf, Germany, Pall’s Acrodisc^® MS Syringe Filter was selected as part of their study into protein secretion by skeletal muscle cells¹. One key challenge of this work was the relatively low abundance of the proteins of interest, necessitating high-performance filtration in order not to adversely affect the experimental results.

It has long been established that muscle and bone share a close functional and developmental relationship. More recent studies have revealed that the muscle secretome, or the collection of peptides and proteins secreted by muscle cells, embodies a communication system that controls bone-muscle interactions, including functions such as growth, development, and aging.²

Analyzing this communication system has proved difficult, however, because high-abundance protein signals often mask low-abundance protein signals, making the discovery of new proteins challenging. The authors of this paper sought to devise a novel methodology that helps circumvent protein signal masking and enables the detection and identification of low-abundance secreted proteins.

A novel method for low-abundance protein detection

The researchers combined a strategy that compares protein abundance between muscle cell lysates and proteins present in muscle cell supernatant (the media in which the cells grow and differentiate). This relative abundance information is then cross-referenced to data derived from a newly developed bioinformatics tool that the researchers call the “Lysate and Secretome Peptide Feature Plotter” (LSPFP). The LSPFP tool is designed to detect protein processing events likely to occur during secretion.

While several secreted protein prediction tools already exist, they are generally restricted to the presence of known signal peptide cleavage sites. The newly developed prediction tool is more comprehensive. It takes classic cleavage sites into account but also searches for several biologically significant protein processing mechanisms by which proteins can be shed from the cell surface, or by which a larger protein may be cleaved to produce smaller bioactive pieces.

To carry out their work, the research team grew muscle cells in culture under conditions that would induce the cells to differentiate into myotubes (primitive muscle fibers) in the same way that they would in the human body. After a few days of growth and differentiation into myotubes, the cultured muscle cells were washed and then incubated for five hours in serum-free medium to minimize potential signal interference by serum proteins. This conditioned medium, referred to as the secretome, was collected for proteomic analysis by mass spectrometry (MS).

The muscle cell myotubes themselves were also harvested, lysed to extract proteins, and prepared for MS analysis. This analysis set was referred to as the cellular proteome.

For both the cellular proteome and secretome analysis sets, samples were collected either with or without Brefeldin A treatment. Brefeldin A is used to block protein transport out of the cell. Control and Brefeldin A treated samples were used as an additional method of cross-checking that identified proteins were being secreted via classical cellular transport mechanisms.

High-performance syringe filtration step

Sample preparation is undoubtedly one of the most important considerations for MS analysis since the method is highly dependent on peptide signal strength and ionization. For this reason, it is important to remove any contaminants that could interfere with the analysis. For research aimed at discovering previously unidentified secreted low-abundance proteins, optimizing MS sample preparation is critical to success. For MS analysis, all cellular proteome and cellular secretome samples were therefore sterile filtered using Pall’s Arodisc MS Syringe Filiters in order to remove any dead cells or cellular debris. 

The Acrodisc MS Syringe filter is the first Liquid chromatography–mass spectrometry (LCMS) certified filter with extremely low levels of extractables, thus minimizing the risk of ion suppression or enhancement which can affect qualitative and quantitative analyses.  The filter features a WWPTFE (water wettable polytetrafluoroethylene) membrane that can be used with both organic and aqueous solvents, with minimal protein absorption. Acrodisc MS Syringe Filters have a 0.2 µm pore size and are available in a 13 mm format with a 49 mL/min flow rate at 30 psi at ambient temperature, or a 25 mm format with a 140 mL/min flow rate at 30 psi at ambient temperature. Following sterile filtration, MS samples were analyzed on a mass spectrometer, and the resulting data was compiled for analysis.

For the first part of their analysis, the scientists used MS sequencing tools to identify those proteins more abundant in the muscle cell secretome versus the proteome. These protein identifications corresponded with an 80% hit rate to proteins identified as secreted via Brefeldin A analysis.

For the second part of their analysis, the researchers used their LSPFP bioinformatics tool to detect protein cleavage events that might occur during secretion. Using this method, several proteins were identified as candidates for protein processing or shedding that would result in their being part of the secretome. The candidates included peptides from plexin-B2, a protein previously confirmed to be processed in this manner.

This research, and the workflows that make it possible, will help scientists learn more about what sort of signaling events take place between muscle and bone, and how to prevent diseases that can result when these signals are disrupted.

Pall is proud to support research workflows with industry-leading products such as the Acrodisc MS Syringe Filters. Learn more about filtration solutions for analytical sample preparation for reliable results and superior column protection.