Investigating the secretome: lessons about the cells that comprise the heart

Abstract

The cell/environment interface is composed of the proteins of plasma membrane which face the extracellular space and by the proteins secreted directly by the cell of origin or by neighboring cells. The secreted proteins can act as extracellular matrix proteins and/or autocrine/paracrine proteins. This report discusses the technical aspects involved in the identification and characterization of the secreted proteins of specific cell types that comprise the heart. These aspects include the culturing of the cells, cell co-culturing and quantitative labeling, conditioned media collection and dealing with high abundant serum proteins, post-translational modification enrichment, the use of protein separation methods and mass spectrometry, protein identification and validation and the incorporation of pathway analysis to better understand the novel discovery on the background of already known experimental biological systems. The proteomic methods have the solid emplacement in cardiovascular research and the identification of proteins secreted by cardiac cells has been used in various applications such as determination the specificity between secretomes of different cell types, e.g. cardiac stem cells and cardiac myocytes, for the global secretome screening of e.g. human arterial smooth muscle cells, for the mapping of the beneficial effect of conditioned medium of one cell type on the other cell type, e.g. conditioned medium of human mesenchymal stem cells on cardiac myocytes, and for the searching the candidate paracrine factors and potential biomarkers.

Figure 1

Depletion of albumin in culture medium containing FBS. A - image of SDS-PAGE gel after silver staining; lane 1 - bovine serum albumin (6 µg loaded), lane 2 - medium containing 2 % FBS before albumin depletion (5 µl loaded is equal to 1.25 µg of total protein), lane 3 - medium containing 2% FBS after albumin depletion (5 µl of flow through loaded). B - chromatograms of RPLC of the medium containing 10 % FBS before albumin depletion (solid arrow; 100 µl loaded is equal to 56 µg of total protein) and after albumin depletion (dashed arrow; 100 µl of flow through loaded). Inset – example of spectrum of one peptide DAFLGSFLYEYSR identifying bovine albumin (accession # P02769; ALBU_BOVIN) present in the FBS culture medium. ProteaPrep Albumin Depletion Sample Prep Kit was from Protea Biosciences (WV, USA; cat. # SP-200-12), albumin was from Sigma (MO, USA; cat. # A7517) and media was Clonetics smooth muscle cell basal medium (Lonza, MD, USA; cat. # CC-3181).

Figure 2

Schematic workflow and MRM analysis of one peptide unique to the target protein ST2. The ST2 protein was digested with trypsin and the resulting peptides were analyzed using a triple quadrupole MS instrument. Peptides were targeted within the MS instrument and only those peptides selected based on their mass (parent ion) were analyzed. The specificity was obtained based on the peptides elution times from the online RPLC and the corresponding MS/MS data (transitions). Three transitions are shown for one selective parent peptide (ITDFGEPR) that is unique for the protein ST2 (accession # Q01638; ILRL1_HUMAN). The MS/MS data provides amino acid sequence information and ensures a high level of specificity. Three peptides and two transitions per peptide are commonly used for quantification.

Figure 3

Validation of ANP by Western blotting as identified by LC-MS/MS. A - ANP identified in NRVM medium and NRVM lysate; amounts of total protein loaded into SDS-PAGE gel were 75 µg and 180 µg, respectively; B - ANP in CSC medium and NRVM medium; amounts of total protein loaded into SDS-PAGE gel were 130 µg and 75 µg, respectively. SDS-PAGE conditions: NuPAGE 4–12% Bis-Tris 1.0 mm thick gel (cat. # NP0321; Invitrogen, CA, USA); MES running buffer; 200 V for 35 minutes [17]. The primary antibody (rabbit anti-rat ANP; Pab A4152-35; US Biological, MA, USA) was used in 1:1,000 dilution and secondary antibody-enzyme conjugate (alkaline phosphatase) was used in 1:10,000 dilution. Recombinant rat ANP (1–28 aa from C-terminus, 3063 Da) was from US Biological (MA, USA; cat. # A4152-05). Detailed explanation is given in the text.