Time-course mass spectrometry data of adipose mesenchymal stem cells acquiring chondrogenic phenotype

Objectives Rabbit adipose mesenchymal stem cells were used for the purpose of studying acquisition of the chondrogenic phenotype over time at 1, 14 and 28 days after in vitro incubation with differentiation media, using nano-liquid chromatography electrospray ionization tandem mass spectrometry analysis. This was part of a preliminary study of the behavior of differentiated adipose stem cells for use in a rabbit model of laryngoplasty. Data description The data comprise .MGF, .RAW, .MZID, and .XLSX, lists of peaks, peptides and proteins identified by nano-flow liquid chromatography electrospray ionization tandem mass spectrometry analysis upon incubation with non-differentiating (ND) or chondrogenic differentiating (CHD) media (ProteomeXchange ID PXD010236). XLSX files contain the following information: day 1 CT (control, N = 3499 proteins), day 14 ND (N = 3106 proteins), day 28 ND (N = 3116 proteins), day 14 CHD (N = 2901 proteins), and day 28 CHD (N = 2876 proteins). Proteins are characterized with respect to their − 10lgP value, percent coverage, number of total as well as unique peptides after trypsin digestion, derivatization method (carbamidomethylation, oxidation, or combined carbamidomethylation + oxidation), average mass, and include a full description.


Objective
Adipose mesenchymal stem cells (ADSCs) are fast gaining popularity for various regenerative medicine applications due to their pluripotency as well as relative use of collection and processing. This has called for increased scrutiny and standardization of protocols involved in tissue collection, differentiation and analysis [1]. While analysis based on DNA or RNA expression has been the preferred method for studying gene expression due to the relatively low cost and flexible, scalable features of microarrays, these analyses do not report actual protein levels. Meanwhile, the cost of protein analysis using nano-flow liquid chromatography electrospray ionization tandem mass spectrometry (nLC-MS/MS) now allows for true protein measurements to be made at the tissue or even single cell level. Our lab's interest lies in developing regenerative medicine application for otorhinolaryngology. Currently, we are gaining expertise with new biologicals to address vocal fold (VF) paralysis, one of the complications that can arise from accidental sectioning of the recurrent laryngeal nerve (RLN) during thyroidectomy. As part of developing a rabbit model for injection medialization laryngoplasty (IML), a surgical procedure involving injection of volume augmenting materials that bring the paralyzed VF proximal to its counterpart, we established protocols for growing and differentiating ADSCs into chondrocytes in vitro. The data now allow for time-course analyses of true protein levels with the potential of revealing new proteins and protein network

Data description
Five lists of proteins were generated by nLC-MS/MS analysis of rabbit ADSC incubated with non-differentiating (ND) or chondrogenic differentiating (CHD) media for 1, 14, or 28 days.  7) No. of Unique Peptides, (8) Derivatization Method, (9) Average Mass, and (10) Description. A Protein Group is a list of proteins sharing a significant number of identical matched peptides. − 10 logP expresses the confidence of making a protein call; high values reflect high confidence [2]. Coverage refers to the percentage of amino acids in the matched peptides relative to the total number of amino acids in the protein.

Chondrogenic differentiation
Chondrogenic differentiation was performed as previously described [3]. Briefly, ADSCs were prepared in 15 mL polystyrene tubes as collagen macrospheres by mixing 5 × 10 5 cells with 50 µL of collagen followed by sedimentation at 500 g for 5 min and drop-wise addition of StemPro Chondrogenic differentiation media (Basal Medium + Chondrogenic Supplement, A10071-01 Life Technologies, Carlsbad, CA). This was supplemented with 5 ng/mL of TGF-β2 (T2815; Sigma-Aldrich, St. Louis, MO). Non-differentiating media consisted of the Basal Medium alone. All media was replenished every 2-3 days. Chondrogenic differentiation was confirmed by Alcian blue staining and sulfated glycosaminoglycan (s-GAG) level analysis with DNA content correction using PicoGreen (Quant-iT P7589, Invitrogen, Carlsbad, CA). Parallel cultures in adipogenic and osteogenic differentiation media (StemPro A1007001 and A1007201; ThermoFisher, Minneapolis, MN) were performed to confirm the presence of pluripotent stem cells in our samples. These data were published elsewhere [3].

nLC-MS/MS analysis
After 1, 14, or 28 days the cell pellets were washed ten times with PBS, fixed in 4% paraformaldehyde and processed for nLc-MS/MS analysis as previously reported [4]. The sample preparation protocol is available on line (  [5]. Only proteins containing two or more unique peptides were compared across groups [6].

Limitations
The data presented here describe acquisition of the chondrogenic phenotype by rabbit ADSCs. The data are useful for the appreciation of time-dependent cellular changes in ADSCs at 14 and 28 days of incubation with chondrogenic differentiation media. The data are also useful for a comparison across species, for example with mouse or human ADSCs, aimed at identifying patterns of similar or differential expression of proteins implicate in acquisition of the chondrogenic phenotype. The main limitations of the data are the lack of replicate measurements and time points beyond day 28. Furthermore, because of the lack of a finer temporal resolution, transient, or intermediary events are not properly captured.