title: Bottom-up proteomic analysis of single HCT 116 colon carcinoma multicellular spheroids.
abstract: BACKGROUND
  Proteomic analysis of single multicellular spheroids has not been previously reported. As three-dimensional cell cultures are an increasingly popular model system for biological research, there is interest in obtaining proteomic profiles of these samples. We investigated the proteome of single HCT 116 multicellular spheroids using protocols optimized for small sample sizes.
  
  
  METHODS
  Six biological replicates were analyzed via microscopy for size. Total protein content was assessed via the bicinchoninic acid assay (BCA assay). Five separate biological replicate spheroids were analyzed via mass spectrometry in technical duplicate. An ultra-performance liquid chromatography (UPLC) system coupled with an LTQ Orbitrap Velos was used for peptide separation, analysis, and identification.
  
  
  RESULTS
  The average diameter of six replicate HCT 116 spheroids was 940 ± 30 µm and the average total protein amount was determined to be 39 ± 4 µg. At least 1300 protein groups were identified in each single LC/MS/MS run with 10% of the material from each single spheroid loaded. Database search results showed variation between spheroid protein group identifications. Pearson correlations show that the disparity in identifications is due to random variations in spectra and protocol.
  
  
  CONCLUSIONS
  We detected more than 1350 protein groups in each replicate HCT 116 spheroid. While some variation was detected between replicates, differences in the number of protein groups identified were determined to be the result of random variations in mass spectra acquisition.
authors.affiliation: 251 Nieuwland Science Hall, Department of Chemistry and Biochemistry and the Harper Cancer Research Institute, University of Notre Dame, Notre Dame, IN, 46556, USA.|Integrated Biomedical Sciences Program, University of Notre Dame, Notre Dame, IN, 46556, USA.
authors.forename: Peter E
authors.initials: PE
authors.lastname: Feist
authors.affiliation: 251 Nieuwland Science Hall, Department of Chemistry and Biochemistry and the Harper Cancer Research Institute, University of Notre Dame, Notre Dame, IN, 46556, USA.
authors.forename: Liangliang
authors.initials: L
authors.lastname: Sun
authors.affiliation: 251 Nieuwland Science Hall, Department of Chemistry and Biochemistry and the Harper Cancer Research Institute, University of Notre Dame, Notre Dame, IN, 46556, USA.
authors.forename: Xin
authors.initials: X
authors.lastname: Liu
authors.affiliation: 251 Nieuwland Science Hall, Department of Chemistry and Biochemistry and the Harper Cancer Research Institute, University of Notre Dame, Notre Dame, IN, 46556, USA.
authors.forename: Norman J
authors.initials: NJ
authors.lastname: Dovichi
authors.affiliation: 251 Nieuwland Science Hall, Department of Chemistry and Biochemistry and the Harper Cancer Research Institute, University of Notre Dame, Notre Dame, IN, 46556, USA.
authors.forename: Amanda B
authors.initials: AB
authors.lastname: Hummon
mesh_terms: D002851:Chromatography, High Pressure Liquid; D045325:HCT116 Cells; D006801:Humans; D020543:Proteome / Q000032:analysis* / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D018874:Spheroids, Cellular / Q000737:chemistry*; D053719:Tandem Mass Spectrometry; D046509:Tissue Culture Techniques; D014407:Tumor Cells, Cultured / Q000737:chemistry*
chemical_list: D020543:Proteome