Abstract：
:Proteomics of human tissues and isolated cellular subpopulations create new opportunities for therapy and monitoring of a patients' treatment in the clinic. Important considerations in such analysis include recovery of adequate amounts of protein for analysis and reproducibility in sample collection. In this study we compared several protocols for proteomic sample preparation: i) filter-aided sample preparation (FASP), ii) in-solution digestion (ISD) and iii) a pressure-assisted digestion (PCT) method. PCT method is known for already a decade [1], however it is not widely used in proteomic research. We assessed protocols for proteome profiling of isolated immune cell subsets and formalin-fixed paraffin embedded (FFPE) tissue samples. Our results show that the ISD method has very good efficiency of protein and peptide identification from the whole proteome, while the FASP method is particularly effective in identification of membrane proteins. Pressure-assisted digestion methods generally provide lower numbers of protein/peptide identifications, but have gained in popularity due to their shorter digestion time making them considerably faster than for ISD or FASP. Furthermore, PCT does not result in substantial sample loss when applied to samples of 50 000 cells. Analysis of FFPE tissues shows comparable results. ISD method similarly yields the highest number of identifications. Furthermore, proteins isolated from FFPE samples show a significant reduction of cleavages at lysine sites due to chemical modifications with formaldehyde-such as methylation (+14 Da) being among the most common. The data we present will be helpful for making decisions about the robust preparation of clinical samples for biomarker discovery and studies on pathomechanisms of various diseases.
journal_name
Talantajournal_title
Talantaauthors
Pirog A,Faktor J,Urban-Wojciuk Z,Kote S,Chruściel E,Arcimowicz Ł,Marek-Trzonkowska N,Vojtesek B,Hupp TR,Al Shboul S,Brennan PM,Smoleński RT,Goodlett DR,Dapic Idoi
10.1016/j.talanta.2021.122568keywords:
["Cellular subpopulations","FASP","FFPE tissue Proteomics","In-solution digestion","Pressure-cycling digestion","Proteomics"]subject
Has Abstractpub_date
2021-10-01 00:00:00pages
122568eissn
0039-9140issn
1873-3573pii
S0039-9140(21)00489-6journal_volume
233pub_type
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