Toward a better analysis of secreted proteins: the example of the myeloid cells secretome

Abstract

The analysis of secreted proteins represents a challenge for current proteomics techniques. Proteins are usually secreted at low concentrations in the culture media, which makes their recovery difficult. In addition, culture media are rich in salts and other compounds interfering with most proteomics techniques, which makes selective precipitation of proteins almost mandatory for a correct subsequent proteomics analysis. Last but not least, the non-secreted proteins liberated in the culture medium upon lysis of a few dead cells heavily contaminate the so-called secreted proteins preparations. Several techniques have been used in the past for concentration of proteins secreted in culture media. These techniques present several drawbacks, such as coprecipitation of salts or poor yields at low protein concentrations. Improved techniques based on carrier-assisted TCA precipitation are described and discussed in this report. These techniques have been used to analyze the secretome of myeloid cells (macrophages, dendritic cells) and enabled to analyze proteins secreted at concentrations close to 1 ng/mL, thereby allowing the detection of some of the cytokines (TNF, IL-12) secreted by the myeloid cells upon activation by bacterial products.

2-Deoxy d-Glucose Prevents Cell Surface Expression of NKG2D Ligands through Inhibition of N-Linked Glycosylation

“…Protein precipitation using TCA was done as described by Chevallet et al (32). The cells in this experiment was incubated in RPMI 1640 media with 0.5% FBS.…”

From Secretome Analysis to Immunology

“…At day 11, the bone marrow cells are differentiated into DCs and ready for the various experiments. The resulting cells are essentially CD8-positive, and their cytokinic profile upon LPS activation is (IL-6/IL-12/TNF␣)/high, (IL-10/interferon ␥)/low (18). In some control experiments, dendritic cells were cultured without FLT3-L, leading to a much lower yield of cells.…”

“…In addition to the well known cytokine expression, which is even a test to check for dendritic cell activation with various stimuli (6,7,14), recent work has also shown that activated dendritic cells secrete many other proteins, such as proteases or complement proteins (18,28).…”

“…Among those, some proteins were not surprising as for example the metalloproteases (e.g. MMP12) (18), which are likely used by the DCs to make their way in and out of the peripheral tissues. However, we were quite surprised to detect some proteins that can be viewed as extracellular sensors, such as lipocalin-2 and the YM1 protein.…”

Understanding dendritic cell biology and its role in immunological disorders through proteomic profiling

“…Thereby only a ''truly'' secreted protein fraction was obtained, as opposed to the supernatant of culture cells which is usually composed of a mixture of secreted proteins, cytoplasmic proteins derived from dying cells and residual serum proteins. This issue was already addressed in an earlier study [85], where it was shown that the protein contamination in the DC culture media is linked to various classes of proteins, including cytoskeletal proteins and major metabolic enzymes. To overcome this problem, protein extracts from serum-depleted DC culture media were compared to extracts of total cell lysates in the media, creating a much more comparable 2-D pattern and allowing the identification of specific and truly secreted proteins by DCs.…”