Miniaturized sample preparation on a digital microfluidics device for sensitive bottom-up microproteomics of mammalian cells using magnetic beads and mass spectrometry-compatible surfactants

Lab on a Chip ◽  
2019 ◽  
Vol 19 (20) ◽  
pp. 3490-3498 ◽  
Author(s):  
Jan Leipert ◽  
Andreas Tholey
Keyword(s):  
Mass Spectrometry ◽  
Sample Preparation ◽  
Mammalian Cells ◽  
Magnetic Beads ◽  
Digital Microfluidics ◽  
Bottom Up ◽  
Polymer Surfactants

The combination of digital microfluidics and magnetic beads for removal of polymer surfactants enables sensitive LC-MS-based microproteomics analyses down to 100 mammalian cells.

scholarly journals Profiling embryonic stem cell differentiation by MALDI TOF mass spectrometry: development of a reproducible and robust sample preparation workflow

The Analyst ◽  
2019 ◽  
Vol 144 (21) ◽  
pp. 6371-6381 ◽  
Author(s):  
Rachel E. Heap ◽  
Anna Segarra-Fas ◽  
Alasdair P. Blain ◽  
Greg M. Findlay ◽  
Matthias Trost
Keyword(s):  
Mass Spectrometry ◽  
Sample Preparation ◽  
Mammalian Cells ◽  
Preparation Method ◽  
Embryonic Stem ◽  
Stem Cell Differentiation ◽  
Embryonic Stem Cell Differentiation ◽  
Sample Preparation Method ◽  
Maldi Tof ◽  
Tof Ms

A fast and robust sample preparation method for MALDI TOF MS analysis of whole mammalian cells and phenotypic profiling of mESCs.

scholarly journals S-Trap eliminates cell culture media polymeric surfactants for effective proteomic analysis of mammalian cell bioreactor supernatants

2020 ◽  
Author(s):  
Lucia F. Zacchi ◽  
Dinora Roche Recinos ◽  
Ellen Otte ◽  
Campbell Aitken ◽  
Tony Hunt ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Cell Culture ◽  
Sample Preparation ◽  
Mammalian Cell ◽  
Proteomic Analysis ◽  
Mammalian Cells ◽  
Culture Media ◽  
Quality Data ◽  
Polymeric Surfactants ◽  
Cell Culture Media

AbstractProteomic analysis of bioreactor supernatants can inform on cellular metabolic status, viability, and productivity, as well as product quality, which can in turn help optimize bioreactor operation. Incubating mammalian cells in bioreactors requires the addition of polymeric surfactants such as Pluronic F68, which reduce the sheer stress caused by agitation. However, these surfactants are incompatible with mass spectrometry proteomics and must be eliminated during sample preparation. Here, we compared four different sample preparation methods to eliminate polymeric surfactants from filtered bioreactor supernatant samples: organic solvent precipitation; filter-assisted sample preparation (FASP); S-Trap; and single-pot, solid-phase, sample preparation (SP3). We found that SP3 and S-Trap substantially reduced or eliminated the polymer(s), but S-Trap provided the most robust clean-up and highest quality data. Additionally, we observed that SP3 sample preparation of our samples and in other published datasets was associated with partial alkylation of cysteines, which could impact the confidence and robustness of protein identification and quantification. Finally, we observed that several commercial mammalian cell culture media and media supplements also contained polymers with similar mass spectrometry profiles, and we suggest that proteomic analyses in these media will also benefit from the use of S-Trap sample preparation.

scholarly journals Histone sample preparation for bottom-up mass spectrometry: a roadmap to informed decisions

2021 ◽  
Author(s):  
Simon Daled ◽  
Sander Willems ◽  
Bart Van Puyvelde ◽  
Laura Corveleyn ◽  
Sigrid Verhelst ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Sample Preparation ◽  
Research Question ◽  
Stable Gene ◽  
Assessment Procedure ◽  
Bottom Up ◽  
Genome Complexity ◽  
Arginine Residues ◽  
Multicellular Organisms ◽  
Selection Of

Histone-based chromatin organization enabled eukaryotic genome complexity. This epigenetic control mechanism allowed for the differentiation of stable gene-expression and thus the very existence of multicellular organisms. This existential role in biology makes histones one of the most complexly modified molecules in the biotic world, which makes these key regulators notoriously hard to analyze. We here provide a roadmap to enable fast and informed selection of a bottom-up mass spectrometry sample preparation protocol that matches a specific research question. We therefore propose a two-step assessment procedure: (i) visualization of the coverage that is attained for a given workflow and (ii) direct alignment-between-runs to assess potential pitfalls at the ion level. To illustrate the applicability, we compare four different sample preparation protocols, while adding a new enzyme to the toolbox, i.e., RgpB (GingisREX, Genovis), an endoproteinase that selectively and efficiently cleaves at the c-terminal end of arginine residues.

scholarly journals Histone Sample Preparation for Bottom-Up Mass Spectrometry: A Roadmap to Informed Decisions

Proteomes ◽  
2021 ◽  
Vol 9 (2) ◽  
pp. 17
Author(s):  
Simon Daled ◽  
Sander Willems ◽  
Bart Van Puyvelde ◽  
Laura Corveleyn ◽  
Sigrid Verhelst ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Sample Preparation ◽  
Research Question ◽  
Stable Gene ◽  
Assessment Procedure ◽  
Bottom Up ◽  
Genome Complexity ◽  
Arginine Residues ◽  
Multicellular Organisms ◽  
Selection Of

Histone-based chromatin organization enabled eukaryotic genome complexity. This epigenetic control mechanism allowed for the differentiation of stable gene-expression and thus the very existence of multicellular organisms. This existential role in biology makes histones one of the most complexly modified molecules in the biotic world, which makes these key regulators notoriously hard to analyze. We here provide a roadmap to enable fast and informed selection of a bottom-up mass spectrometry sample preparation protocol that matches a specific research question. We therefore propose a two-step assessment procedure: (i) visualization of the coverage that is attained for a given workflow and (ii) direct alignment between runs to assess potential pitfalls at the ion level. To illustrate the applicability, we compare four different sample preparation protocols while adding a new enzyme to the toolbox, i.e., RgpB (GingisREX®, Genovis, Lund, Sweden), an endoproteinase that selectively and efficiently cleaves at the c-terminal end of arginine residues. Raw data are available via ProteomeXchange with identifier PXD024423.

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