Proteomics

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Towards an optimized workflow for middle-down proteomics

ABSTRACT: Detailed description of an optimally tuned workflow for high throughput middle-down proteomics with the currently available state-of-the-art instrumentation. In this work we describe and critically evaluate the limits of an optimized workflow for the detection of middle-range peptides. First, the work compares the yield of desired longer peptides both by enzymatic and acidic digestion protocols. Second, it uses optimized conditions for sample clean-up and multidimensional LC. Third, we fine-tuned critical MS parameters for improving the detection of middle-range peptides where we comprehensively evaluated ETD, HCD and EThcD fragmentation schemes on all the digests obtained in order to choose the best sequencing method for distinct populations of peptides and with special attention paid to longer peptides.

INSTRUMENT(S): Orbitrap Fusion ETD

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Alba Cristobal  

LAB HEAD: Albert J.R. Heck

PROVIDER: PXD004910 | Pride | 2017-03-16

REPOSITORIES: Pride

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Dataset's files

Source:
Action DRS
20160127_FMABC_zorbax300_AspN_ETD_SCX_Fr.msf Msf
20160127_FMABC_zorbax300_AspN_EThcD_SCX_Fr.msf Msf
20160127_FMABC_zorbax300_AspN_EThcD_charge_SCX.msf Msf
20160204_FMABC_zorbax300_GluC_EThcD_Fr.msf Msf
20160204_FMABC_zorbax300_GluC_HCD_Fr.msf Msf
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Toward an Optimized Workflow for Middle-Down Proteomics.

Cristobal Alba A   Marino Fabio F   Post Harm H   van den Toorn Henk W P HW   Mohammed Shabaz S   Heck Albert J R AJ  

Analytical chemistry 20170310 6

Mass spectrometry (MS)-based proteomics workflows can crudely be classified into two distinct regimes, targeting either relatively small peptides (i.e., 0.7 kDa < M<sub>w</sub> < 3.0 kDa) or small to medium sized intact proteins (i.e., 10 kDa < M<sub>w</sub> < 30 kDa), respectively, termed bottom-up and top-down proteomics. Recently, a niche has started to be explored covering the analysis of middle-range peptides (i.e., 3.0 kDa < M<sub>w</sub> < 10 kDa), aptly termed middle-down proteomics. Alt  ...[more]

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