Proteomics

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Thiol reactive tandem mass tags for quantitative top-down proteomics

ABSTRACT: Here, we describe the development of a labeling strategy for TDP targeting thiol groups of cysteine residues with iodoTMTsixplex. While this method inherently excludes the quantification of cysteine-free proteoforms, it provides the opportunity of sixplexing and the implementation of multidimensional separation schemes. The approach was tested using both a high/low-pH RP-LC and a gel-eluted liquid fraction entrapment electrophoresis (GelFrEE) x RP-LC separation. Over- and underlabeling rates were determined and MS/MS parameters were optimized to enable parallel protein identifications and quantification. Finally, a complex two proteome interference model was applied to demonstrate the high accuracy of the developed quantification method.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Escherichia Coli Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Andreas Tholey  

LAB HEAD: Andreas Tholey

PROVIDER: PXD026310 | Pride | 2021-08-06

REPOSITORIES: Pride

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1ug_30kDa_Batch1_EThcD25_HCD80_2x118ms.raw Raw
1ug_30kDa_Batch1_EThcD25_HCD80_2x118ms_20210220183116.raw Raw
1ug_30kDa_Batch1_EThcD25_HCD80_2x22ms.raw Raw
1ug_30kDa_Batch1_EThcD25_HCD80_2x22ms_20210220040917.raw Raw
1ug_30kDa_Batch1_EThcD25_HCD80_2x246ms.raw Raw
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Publications

Quantitative Top-Down Proteomics by Isobaric Labeling with Thiol-Directed Tandem Mass Tags.

Winkels Konrad K   Koudelka Tomas T   Tholey Andreas A  

Journal of proteome research 20210802 9

While identification-centric (qualitative) top-down proteomics (TDP) has seen rapid progress in the recent past, the quantification of intact proteoforms within complex proteomes is still challenging. The by far mostly applied approach is label-free quantification, which, however, provides limited multiplexing capacity, and its use in combination with multidimensional separation is encountered with a number of problems. Isobaric labeling, which is a standard quantification approach in bottom-up  ...[more]

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