Here we have developed and validated an original LC-MS/MS SRM procedure flexible enough to quantitatively screen collagen types I-V in copies of the same type of stromal matrix prepared with different protocols of cell removal to retain the native 3D architecture of the ECM. In a first step, identification of tryptic sequences exclusive to specific chains (either α1 or α2) of mammalian collagen standards types I-V was pursued using a combination of LC-LIT-Orbitrap XL and LC-MS/MS SRM analyses. In a second step, the adult male rat thyroid was decellularized using three different protocols specifically set for engineering of bioartificial 3D thyroid organoids. In a third step, DNA analysis of the decellularized 3D thyroid stroma was pursued to exclude contamination by cell nuclear debris. In a final step, collagen standards and 3D thyroid matrices were digested using the same mechanical / enzymatic protocol, and quantitative profiles of collagen types I-V ensued using comparisons of ionic intensities between tryptic peptides of collagen standards and matrices, as derived from targeted LC-MS/MS SRM analysis. Collectively, the procedure allowed for detection and quantitation of collagen types I-V at a femtomolar level in thyroid gland stromal matrices initially maintaining their original 3D architecture, tryptically digested through a method common to collagen standards and thyroid ECM, with satisfactory reproducibility of results, moderate procedural cost, and limited analytical time.

A targeted mass spectrometry method to screen collagen types I-V in the decellularized 3D extracellular matrix of the adult male rat thyroid / Alfieri, M; Barbaro, F; Consolini, Elia; Bassi, E; Dallatana, D; Bergonzi, C; Bianchera, A; Bettini, R; Toni, R; Elviri, L.. - In: TALANTA. - ISSN 0039-9140. - 193:(2019), pp. 1-8. [10.1016/j.talanta.2018.09.087]

A targeted mass spectrometry method to screen collagen types I-V in the decellularized 3D extracellular matrix of the adult male rat thyroid

Barbaro F;CONSOLINI, ELIA;Dallatana D;Bergonzi C;Bianchera A;Bettini R;Toni R;Elviri L.
2019

Abstract

Here we have developed and validated an original LC-MS/MS SRM procedure flexible enough to quantitatively screen collagen types I-V in copies of the same type of stromal matrix prepared with different protocols of cell removal to retain the native 3D architecture of the ECM. In a first step, identification of tryptic sequences exclusive to specific chains (either α1 or α2) of mammalian collagen standards types I-V was pursued using a combination of LC-LIT-Orbitrap XL and LC-MS/MS SRM analyses. In a second step, the adult male rat thyroid was decellularized using three different protocols specifically set for engineering of bioartificial 3D thyroid organoids. In a third step, DNA analysis of the decellularized 3D thyroid stroma was pursued to exclude contamination by cell nuclear debris. In a final step, collagen standards and 3D thyroid matrices were digested using the same mechanical / enzymatic protocol, and quantitative profiles of collagen types I-V ensued using comparisons of ionic intensities between tryptic peptides of collagen standards and matrices, as derived from targeted LC-MS/MS SRM analysis. Collectively, the procedure allowed for detection and quantitation of collagen types I-V at a femtomolar level in thyroid gland stromal matrices initially maintaining their original 3D architecture, tryptically digested through a method common to collagen standards and thyroid ECM, with satisfactory reproducibility of results, moderate procedural cost, and limited analytical time.
A targeted mass spectrometry method to screen collagen types I-V in the decellularized 3D extracellular matrix of the adult male rat thyroid / Alfieri, M; Barbaro, F; Consolini, Elia; Bassi, E; Dallatana, D; Bergonzi, C; Bianchera, A; Bettini, R; Toni, R; Elviri, L.. - In: TALANTA. - ISSN 0039-9140. - 193:(2019), pp. 1-8. [10.1016/j.talanta.2018.09.087]
File in questo prodotto:
Non ci sono file associati a questo prodotto.

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11381/2855983
Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus 3
  • ???jsp.display-item.citation.isi??? 3
social impact