Developing a methodology for the collection and analysis of cell secretomes

Studying the secreted proteome (or ‘secretome’) from cultured cells presents several challenges. These relate to the contamination of the pool of secreted proteins by intracellular proteins released from dead or dying cells, the low concentration of secreted protein within the culture medium and the masking of these proteins by serum proteins (e.g. BSA) used in most cell culture media [632]. Thus, experiments were carried out to establish methodologies that address these potential problems.

To avoid the effects of a large amount of contaminating serum proteins in both LC- MS and gel electrophoresis analyses of the secretomes, cells were incubated in serum-free Rama 37 medium, and the resulting conditioned medium (CM) was collected for analysis by LC-MS (see Materials and Methods). A total of 802 proteins were identified in the comparative CMs of WT AGR2 pool and EV pool cells (including differentially and non- differentially expressed proteins), and these proteins were subjected to further analysis using the Ingenuity Pathway Analysis (IPA) software package (Qiagen). Of the proteins identified, 627 (78.2 %) could be mapped to the IPA database, and information on the recorded subcellular localisation of these proteins could be collected for 527 (90.1 % mapped proteins, 71.1 % total proteins) of these (Fig. 5.1).

300, 52% 101, 18% 81, 14% 73, 13% 15, 3% Cytoplasm Extracellular space Plasma membrane Nucleus Other

Figure 5.1. Distribution of proteins between intracellular and extracellular compartments from conditioned medium of WT AGR2 pool and EV pool cells incubated in serum-free DMEM medium. CM proteins were analysed by LC-MS and their cellular distribution determined using Ingenuity Pathway Analysis. Note that this encompasses all proteins identified in the CM of both cell types, not just differentially expressed proteins.

Chapter 5 Results

156 Only 101 (18 %) of the identified proteins were listed as belonging to the extracellular space, with 81 (14 %) proteins labelled as membrane proteins. Given that one proteomic study identified 18 of 22 transmembrane proteins from human mammary epithelial cells as having an extracellular shed form [633], at least some of the membrane proteins identified in the WT AGR2 pool and EV pool secretomes could also be shed- domains of membrane proteins. However, 373 (65 %) of the secretome proteins were of intracellular origin (cytoplasmic or nuclear), suggesting that there was substantial contamination of the secretome by intracellular proteins, presumably released from dead and dying cells.

Several studies investigating secreted proteins have used Opti-MEM serum-free medium (Gibco) for the collection of these proteins [634-638]. Opti-MEM is a chemically- defined, low protein concentration medium containing growth factors to support growth in the absence of FBS. To try and minimise the contamination of the secretome by intracellular proteins observed with serum-free DMEM medium, EV and WT AGR2 pool cells were incubated in Opti-MEM medium and the CM again analysed by LC-MS and IPA (Fig. 5.2).

A similar number of proteins were identified in the CM of Opti-MEM-incubated cells (783 proteins), and both the identity of these proteins and the overall distribution of proteins between cellular compartments was largely the same as from DMEM-incubated

296, 51% 117, 20% 103, 17% 58, 10% 10, 2% Cytoplasm Extracellular Space Plasma membrane Nucleus Other

Figure 5.2. Distribution of proteins between intracellular and extracellular compartments from conditioned medium of WT AGR2 pool and EV pool cells incubated in serum-free Opti-MEM medium. CM proteins were analysed by LC-MS and their cellular distribution determined using Ingenuity Pathway Analysis. Note that this encompasses all proteins identified in the CM of both cell types, not just differentially expressed proteins.

Chapter 5 Results

157 cells. However, the overall concentrations of these proteins in the CMs, based on peptide abundances calculated by Progenesis software (see Materials and Methods), differed between DMEM- and Opti-MEM-incubated cells. This suggests that simply examining all the proteins identified in CMs may not be an appropriate method for determining the suitability of the CM collection method, and that the relative abundances of these proteins should be taken into account. This necessitates the use of a normalisation factor to compare protein abundances across different cell lines and CM collection methods. Therefore, one bona fide secreted protein, fibronectin [639, 640], was identified in both experiments shown in Fig. 5.1 and 5.2 that showed no significant difference in expression between EV or WT AGR2 cells, and could potentially be used as a normalisation factor for the levels of secreted proteins between WT AGR2 and EV pool cells. In addition, pilot data suggested that there was indeed no difference in the amount of fibronectin secreted from WT AGR2 and EV pool cells, as CM from both cell types analysed by SDS-PAGE showed comparable levels of fibronectin (Fig. 4.8). Similarly, there was no significant difference in intracellular levels of fibronectin between these cells (fold change: 1.12, p = 0.078, ANOVA). This suggests that the secreted levels of fibronectin could be used as a normalisation factor for the secretomes of WT AGR2 and EV pool cells.

With this in mind, the levels of proteins identified in CM collected from DMEM- and Opti-MEM-based serum-free media by LC-MS were normalised to the level of fibronectin in the CMs. The mean level of fibronectin across the four replicate samples for each cell type in each serum-free medium was used as a normalisation factor for the mean levels of each protein identified in the CM of each cell type. This yielded a fibronectin-normalised abundance value that was used to compare the abundances of CM proteins across cell types and incubation media. To determine the most appropriate collection method, the cellular localisation of the most abundant proteins in both collection media was assessed. The most abundant proteins were determined as those present at a level greater than 0.5 times that of fibronectin, retaining 168 proteins from DMEM-incubated cells and 15 from Opti-MEM incubated cells. In essence, the value itself of the cutoff point is not important, as long as it selects for the most abundant proteins; choosing a lower cutoff will simply increase the proportion of proteins included in the analysis that are released as a consequence of cell lysis, which should be present at a lower concentration than true secreted proteins.

Chapter 5 Results

158 Considering only the most abundant proteins, the proportion of proteins denoted as belonging to the extracellular space increased in DMEM-incubated cells rose from 18 % of all CM-derived proteins (Fig. 5.1) to 30 % of the most abundant proteins (Fig. 5.3A). However, 50 % of the most abundant proteins were still classified as cytoplasmic. In the CM from Opti-MEM-incubated WT AGR2 and EV pool cells, only 15 proteins out of all identified proteins from both cell types (i.e. including differentially and non-differentially expressed

73, 43% 50, 30% 29, 17% 11, 7% 5, 3% Cytoplasm Extracellular space Plasma membrane Nucleus Other 13, 87% 2, 13% 0, 0% Extracellular space Cytoplasm

Figure 5.3. Comparison of distribution between intracellular and extracellular compartments of the most abundant proteins from conditioned medium of DMEM- and Opti-MEM-incubated WT AGR2 and EV pool cells. CM proteins were analysed by LC-MS and their cellular distribution determined using Ingenuity Pathway Analysis. Protein abundance was normalised to fibronectin, and the cellular localisation for most abundant proteins (present at a level ≥ 0.5 times the abundance of fibronectin) were determined using Ingenuity Pathway Analysis. (A) DMEM-incubated cells and (B) Opti-MEM-incubated cells.

A

Chapter 5 Results

159 proteins) were found at levels ≥ 0.5 times that of fibronectin (Fig. 5.3B). Of these, only 2 (13 %) were not classified as belonging to the extracellular space. Of these two cytoplasmic proteins, one (nucleobindin-1) is resident in the Golgi and the other (SRPRB) is an ER protein. Given the previously observed secretion of other ER proteins [448, 449, 620, 621], these could potentially also be secreted proteins, but overall, this shows that the majority of the most abundant proteins in the CM from Opti-MEM medium are indeed extracellular proteins. These observed differences in the proportion of extracellular and intracellular proteins with these two serum-free media indicate that Opti-MEM is the more suitable method for minimising contamination from intracellular proteins. While CM from these Opti-MEM-incubated cells contained many fewer proteins expressed at ≥ 0.5 times the level of fibronectin, this is not surprising given the generally low abundance of secreted proteins [632]. An independent validation of the method can also be seen in the levels of the cytoplasmic protein lactate dehydrogenase A (LDHA) relative to fibronectin in the extracellular space. Extracellular LDHA is routinely used as a measure of cell lysis [641, 642], and the average levels of LDHA relative to fibronectin in the CM from both EV and WT AGR2 cells were 0.12 for cells incubated in Opti-MEM and 8.44 for cells incubated in DMEM, a 70- fold difference in the relative amount of extracellular LDHA between these methods. This indicates a much higher level of cell lysis in DMEM-incubated cells and reinforces the suitability of Opti-MEM as the optimal medium for collecting CM.