Label-free protein profiling of adipose-derived human stem cells under hyperosmotic treatment

Abstract

Our previous work suggested that treatment of cells with hyperosmotic media during 2D passaging primes cells for cartilage tissue engineering applications. Here, we used label-free proteomic profiling to evaluate the effects of control and hyperosmotic treatment environments on the phenotype of multipotent adipose-derived stem cells (ASCs) cultivated with a chondrogenic growth factor cocktail. Spectra were recorded in a data-independent fashion at alternate low (precursor) and high (product) fragmentation voltages (MS(E)). This method was supplemented with data mining of accurate mass and retention time matches in precursor ion spectra across the experiment. The results indicated a complex cellular response to osmotic treatment, with a number of proteins differentially expressed between control and treated cell groups. The roles of some of these proteins have been documented in the literature as characteristic of the physiological states studied, especially aldose reductase (osmotic stress). This protein acted as a positive control in this work, providing independent corroborative validation. Other proteins, including 5'-nucleotidase and transgelin, have been previously linked to cell differentiation state. This study demonstrates that label-free profiling can serve as a useful tool in characterizing cellular responses to chondrogenic treatment regimes, recommending its use in optimization of cell priming protocols for cartilage tissue engineering.

Figure 1

Principal component analysis of Z-score transformed intensity data processed by the Elucidator program for all 18 LC/MS Chromatograms in this experiment. Each data point represents a chromatogram. Data from control cells grown in 300 mOsM media are all found on the low end of the first principal component axis. Data from the 400 mOsM grown cells (treated) are grouped on the higher end of that axis, suggesting an overall global effect of the osmolarity treatment. Replicate LC/MS runs (of like color) are relatively close together in most cases indicating excellent reproducibility of chromatography and mass spectrometry.

Figure 2

Agglomerative hierarchical cluster of Z-score transformed intensity data processed by the Elucidator program for all 18 LC/MS chromatograms in this experiment. Proteins identified in each chromatogram are labeled as control (300 mOsM) or treated (400 mOsM). Groups of three identically-colored boxes above the results of each LC/MS run indicate the results for three replicate LC/MS runs for each biological replicate (three biological replicates of control (300 mOsM) or treated (400 mOsM) cells). Z-score coloration indicates protein abundance in the sample (red indicates higher abundance, green indicates lower abundance and black equal abundance). The cluster suggests many proteins were downregulated at 400 mOsM (upper part of cluster) and many proteins upregulated at 400 mOsM (lower part of cluster). It should be emphasized that there was no filtering applied for statistical significance, fold change or the relative reliability of the protein identification. Z-score transformation as presented emphasizes small differences.

Figure 3

Example relative quantification data and supporting identification data for a protein of equal unchanged abundance (upper) panel, a protein with increased abundance (middle panel) and one with decreased abundance (lower panel) as a result of treatment (400 mOsM). Relative abundance (%) plots show the results for three replicate LC/MS runs for each biological replicate (three biological replicates of control (300 mOsM) or treated (400 mOsM). For each panel, example graphics for two peptides are shown, including a single component of an isotopic cluster (feature plotted at relative % abundance) generated by the Elucidator program (charge state indicated), and a derived ms/ms spectrum generated by the PLGS program from high collision energy scan of MS^E data. Red peaks are y-ions, blue peaks are b-ions, and green are modified ions (e.g. loss of NH₃) in the MS^E scans. The feature peak in each case is an overlay of aligned MS spectra (the most intense from an isotopic cluster representing a single charge state) of matching m/z and retention time for the 18 LC/MS runs in the experiment. Below the graphics is a table representing relative expression of these proteins in (millions of) relative units. Nine replicates for control and treated are shown. They are ordered sequentially in groups of three representing replicate LC/MS runs of the same biological replicate sample. Glyceraldehyde-3-phosphate dehydrogenase had a ratio of treated/control 1.0 (P = n.s.), ALDR had an abundance ratio of 3.5 (P < 10⁻⁴⁵) and 5NTD had an abundance ratio of 0.62 (P < 10⁻⁴¹). Abundance ratio P-values were calculated within Elucidator as described previously.

Figure 4

Expression data for selected proteins in agglomerative hierarchical cluster of Z-score transformed intensity data processed by the Elucidator program for all 18 LC/MS chromatograms in this experiment. These proteins have at least 1.5-fold response to the treatment, and are represented by at least two peptides for both identification and quantification at ratio P-values as calculated by Elucidator. See text for other acceptance criteria. Mean relative abundance of each protein in control and treated as well as abundance ratio (as calculated by Elucidator) are listed (see Supporting Data Table 1 for individual abundance intensities for each replicate and each protein). Cluster coloration indicates protein abundance in the sample (red indicates higher abundance, green indicates lower abundance and black, unchanged abundance). Some proteins were upregulated at 400 mOsM (upper part of cluster) and some were downregulated at 400 mOsM (lower part of cluster). See Supporting Data Figure 1 for graphic that includes all proteins in an agglomerative hierarchical cluster of Z-score transformed intensity data processed by the Elucidator program. Elucidator identified fibronectin as the (shorter) isoform 3, based on the peptides actually detected (the algorithm seeks to provide the simplest interpretation of the data). However, no spectra were annotated to any unique peptides ascribed to isoform 3, thus the protein is listed here as the canonical isoform (P02751).