Identification of secreted proteins by comparison of protein abundance in conditioned media and cell lysates

Abstract

Analysis of the full spectrum of secreted proteins in cell culture is complicated by leakage of intracellular proteins from damaged cells. To address this issue, we compared the abundance of individual proteins between the cell lysate and the conditioned medium, reasoning that secreted proteins should be relatively more abundant in the conditioned medium. Marked enrichment for signal-peptide-bearing proteins with increasing conditioned media to cell lysate ratio, as well loss of this signal following brefeldin A treatment, confirmed the sensitivity and specificity of this approach. The subset of proteins demonstrating increased conditioned media to cell lysate ratio in the presence of Brefeldin A identified candidates for unconventional secretion via a pathway independent of ER to Golgi trafficking.

Fig. 1.. Comparison of protein abundance in the cell lysate and conditioned media enables identification of secreted proteins.

(A) Experimental approach: Proteins isolated from cell lysates and conditioned media were labeled with tandem-mass-tag (TMT) reagents prior to LC-MS/MS analysis to quantify relative protein abundance. A media/lysate (M/L) abundance ratio was calculated for all proteins identified in both fractions. (B) Venn diagram showing the overlap in proteins detected in the cell lysates and conditioned media. The number of proteins with signal peptides present in each fraction is indicated in brackets. (C) The abundance of known cytoplasmic proteins (ACTB, LDHA, TUBA1B) and well documented secreted proteins (SERPINB1, APOB, PCSK9) in the lysate and media fraction, and their M/L ratio. (D) Proteins were binned into discreet M/L ratio intervals, and the percentage of proteins with signal peptides in each bin was calculated. The number of proteins present in each bin is indicated. (E) Gene ontology (GO) analysis for molecular functions for the set of proteins with M/L ratio greater than 10.

Fig. 2.. M/L ratio analysis combined with BFA treatment enables high-sensitivity identification of conventionally secreted proteins.

(A) Proteins bearing signal peptides are co-translationally inserted into the ER and utilize the conventional ER-Golgi-Secretory vesicle/tubule route (pathway (1)) to exit the cell or to reach other intracellular destinations. The process of ER-Golgi transport is blocked by BFA. A number of cytoplasmic proteins are known to be secreted unconventionally via ER-Golgi independent secretory routes by either translocating through plasma membrane pores that are formed by the secreted protein itself, or by other pore-forming proteins (pathway(2)), or by entering membrane bound organelles that fuse with the plasma membrane (pathway (3)). These latter 2 groups of proteins should be resistant to BFA. (B) Immunoblot analysis of PCSK9 abundance in the Huh7 cell lysate and conditioned media following treatment with 1 μg/ml BFA. Four biological replicates are labeled Rep1–4. (C) Chemiluminescence-based quantification of PCSK9 abundance in Huh7 cell lysates and conditioned media following immunoblotting. BFA treated samples were normalized to controls. (D-G) Volcano plots [47] showing changes to the abundance of proteins in the cell lysate (D) and conditioned media (E), and changes to M/L ratio (F) following BFA treatment measured by TMT-based mass spectrometry. The l2fc and statistical significance are plotted on the x and y-axis, respectively. Signal peptide containing proteins are indicated in green, and non-signal peptide containing in purple. (G) Comparison of l2fc following BFA treatment for individual proteins comparing protein abundance in the media alone (squares) to the M/L ratio (circles).

Fig. 3.. Candidate proteins secreted via unconventional secretory pathways.

(A) M/L ratio of control and BFA treated cells are plotted on the x and y-axis, respectively. The inset shows proteins with a M/L ratio ranging from 0 to 30. Proteins sensitive to BFA have lower M/L ratios relative to untreated controls. The top 10 signal-peptide lacking proteins with the highest M/L ratios (candidate unconventionally secreted proteins) are identified by gene name. (B) M/L ratio of candidate unconventionally secreted proteins and sensitivity to BFA. * indicates an adjusted p value < 0.05, calculated using the limma statistical package.

Fig. 4.. BFA sensitivity of transmembrane domain containing proteins.

Transmembrane domain containing proteins detected in both the media and lysate fractions with a M/L ratio >1. The inset shows a magnified view of the 0–15 M/L ratio range. * indicates an adjusted p value < 0.05, calculated using the limma statistical package.