Abstract
Recently we introduced the concept of Suspension Trapping (STrap) for bottom-up proteomics sample processing that is based upon SDS-mediated protein extraction, swift detergent removal and rapid reactor-type protein digestion in a quartz depth filter trap. As the depth filter surface is made of silica, it is readily modifiable with various functional groups using the silane coupling chemistries. Thus, during the digest, peptides possessing specific features could be targeted for enrichment by the functionalized depth filter material while non-targeted peptides could be collected as an unbound distinct fraction after the digest. In the example presented here the quartz depth filter surface is functionalized with the pyridyldithiol group therefore enabling reversible in-situ capture of the cysteine-containing peptides generated during the STrap-based digest. The described C-STrap method retains all advantages of the original STrap methodology and provides robust foundation for the conception of the targeted in-situ peptide fractionation in the STrap format for bottom-up proteomics. The presented data support the method’s use in qualitative and semi-quantitative proteomics experiments.
Highlights
Current state-of-the-art mass spectrometry (MS) instrumentation employed in proteomics handles a ‘mere’ four orders of magnitude of the protein dynamic range, compared with the no less than seven-orders-of-magnitude spanning protein abundances in living organisms [1]
The MK360 Munktell quartz filter is modified with pyridyldithiol, i.e. a MK360-50 mm filter disk is incubated with 10 ml of 2% (3-Aminopropyl)trimethoxysilane (APTMS) solution in acetone for 2 hours and washed several times with acetone. 7 mg of N-succinimidyl 3(2-pyridyldithiol) propionate (SPDP) is dissolved in 0.5 ml of dimethyl sulfoxide (DMSO) and added into 9.5 ml of the phosphate buffered saline (PBS)/EDTA solution (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4/15 mM EDTA)
The implementation of the C-Suspension Trapping (STrap) technique is proposed in two ways
Summary
Current state-of-the-art mass spectrometry (MS) instrumentation employed in proteomics handles a ‘mere’ four orders of magnitude of the protein dynamic range, compared with the no less than seven-orders-of-magnitude spanning protein abundances in living organisms [1]. The basic principle underlying the STrap methodology is creation of fine particles in suspension from SDS-solubilized proteins, capture of the particles in a quartz depth filter with subsequent in-situ digestion by an introduced protease. During the digestion process, the peptides possessing the targeted functionalities are selectively enriched by the modified depth filter material.
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