Unraveling Mesenchymal Stem Cells’ Dynamic Secretome Through Nontargeted Proteomics Profiling

Abstract

The modulatory and regenerative potential shown by the use of MSC secretomes has emphasized the importance of their proteomics profiling. Proteomic analysis, initially focused on the targeted analysis of some candidate proteins or the identification of the secreted proteins, has been changing to an untargeted profiling also based on the quantitative evaluation of the secreted proteins.The study of the secretome can be accomplished through several different proteomics-based approaches; however this analysis must overcome one key challenge of secretome analysis: the low amount of secreted proteins and usually their high dilution.In this chapter, a general workflow for the untargeted proteomic profile of MSC's secretome is presented, in combination with a comprehensive description of the major techniques/procedures that can be used. Special focus is given to the main procedures to obtain the secreted proteins, from secretome concentration by ultrafiltration to protein precipitation. Lastly, different proteomics-based approaches are presented, emphasizing alternative digestion techniques and available mass spectrometry-based quantitative methods.

Bursaphelenchus xylophilus and B. mucronatus secretomes: a comparative proteomic analysis

“…Secretomes previously concentrated were precipitated with Trichloroacetic acid (TCA) - Acetone45. The protein pellets were ressuspended in 40 μL of SDS-Sample buffer without bromophenol blue and glycerol46, aided by ultrasonication and denaturation at 95 °C.…”

Secretome of Undifferentiated Neural Progenitor Cells Induces Histological and Motor Improvements in a Rat Model of Parkinson's Disease

“…STEM CELLS TRANSLATIONAL MEDICINE published by Wiley Periodicals, Inc. on behalf of AlphaMed Press GE Healthcare, Little Chalfont, U.K., http://www3.gehealthcare.com/en/global_gateway) by ultracentrifugation at 3,000 g during 45 min, as previously described [40]. Afterwards, the secreted proteins, were precipitated with Trichloroacetic acid (TCA)-Acetone method [40] and the washed pellets were resuspended in 40 μL ×2 Laemmli buffer (BioRad, Hercules, CA, http://www.bio-rad.com/), aided by ultrasonication and denaturation at 95 C. Prior to protein digestion, 10 μL of each replicate were used to create a pooled sample for protein identification. After denaturation, samples were alkylated with acrylamide and subjected in gel digestion by using the short-GeLC approach [41], and the formed peptides were desalted using OMIX tips with C18 stationary phase (Agilent Technologies, Glostrup, Denmark, http://www.agilent.com) before liquid chromatographytandem mass spectrometry (LC-MS/MS).…”

A generic normalization method for proper quantification in untargeted proteomics screening

“…HeLa cells were seeded at 12⨯10 3 cell/cm 2 in 55 cm 2 plates (Corning) in a total of 4 plates per condition 352 for mass spectrometry analysis or 1 plate for immunoblotting experiments. After 48 hours in culture 353 (37 ᵒC with 5% of CO2/95 % air and 95% humidity) the culture medium (DMEM medium with 10 % FBS) 354 was discarded and cells were washed twice with warm PBS to remove the remaining FBS [20]. Then, 355 the medium was changed for DMEM medium without FBS and cells were left in culture for 24 or 48 h 356 (control conditions) or, cells were treated with 1 mM of H2O2 for 40 min to promote oxidative stress 357 and then left in DMEM without FBS for 24 h. 358 After 24 h (ctrl and stress conditions) or 48 h of media conditioning, the medium was collected, 359 centrifuged at 290 xg, for 5 min at 4 °C to remove cell debris, and then concentrated using cut-off filters 360 of 5 kDa (Vivaspin20, Sartorius) [20].…”

“…After 48 hours in culture 353 (37 ᵒC with 5% of CO2/95 % air and 95% humidity) the culture medium (DMEM medium with 10 % FBS) 354 was discarded and cells were washed twice with warm PBS to remove the remaining FBS [20]. Then, 355 the medium was changed for DMEM medium without FBS and cells were left in culture for 24 or 48 h 356 (control conditions) or, cells were treated with 1 mM of H2O2 for 40 min to promote oxidative stress 357 and then left in DMEM without FBS for 24 h. 358 After 24 h (ctrl and stress conditions) or 48 h of media conditioning, the medium was collected, 359 centrifuged at 290 xg, for 5 min at 4 °C to remove cell debris, and then concentrated using cut-off filters 360 of 5 kDa (Vivaspin20, Sartorius) [20]. The concentrated conditioned media were precipitated using 361 Trichloroacetic acid (TCA) -Acetone as described in Manadas et al, [21] and the protein pellets were 362 re-suspended in 2× SDS Laemmli buffer.…”