Optimizing the enrichment of cross-linked products for mass spectrometric protein analysis.
BACKGROUND
Chemical cross-linking in combination with a mass spectrometric analysis of the created cross-linked products is an area of growing interest for deriving low-resolution structural information of proteins and protein complexes. One of the greatest challenges is the complexity of the created cross-linking mixtures, which can be met by a charge-based enrichment of cross-linked peptides after proteolytic digestion using strong cation-exchange (SCX) chromatography.


METHODS
SCX chromatography was used for the enrichment of cross-linked peptides with the N-hydroxysuccinimide ester bis(sulfosuccinimidyl)succinate (BS(3)) prior to a mass spectrometric analysis by nano-HPLC/nano-ESI-LTQ-Orbitrap-MS/MS. Bovine serum albumin (BSA) and glutathione S-transferase (GST) were employed as model proteins.


RESULTS
Conditions for SCX enrichment were optimized for obtaining as many interpeptide cross-linked peptides as possible in order to maximize the amount of structural information from a single experiment. With an SCX-based enrichment step of cross-linked products within BSA using the cross-linker BS(3), 154 interpeptidal cross-linking products were identified during nano-HPLC/nano-ESI-MS/MS analyses, whereas analyses without a prior SCX enrichment allowed the identification of merely 20 cross-linked products. The application of the SCX enrichment strategy for the analysis of cross-linked products of GST with BS(3) allowed the identification of 26 interpeptidal cross-linked products compared with 16 without SCX enrichment.


CONCLUSIONS
For both proteins investigated herein, BSA and GST, the introduction of an SCX-based enrichment step prior to nano-HPLC/nano-ESI-MS/MS of cross-linked products led to a considerable gain in structural information.
Department of Pharmaceutical Chemistry & Bioanalytics, Institute of Pharmacy, Martin-Luther University Halle-Wittenberg, Wolfgang-Langenbeck-Str. 4, D-06120, Halle (Saale), Germany.
Romy
R
Fritzsche
Christian H
CH
Ihling
Michael
M
Götze
Andrea
A
Sinz
D000818:Animals; D002417:Cattle; D002852:Chromatography, Ion Exchange / Q000379:methods*; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000302:isolation & purification*; D004926:Escherichia coli / Q000201:enzymology; D005982:Glutathione Transferase / Q000737:chemistry / Q000302:isolation & purification*; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification*; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000302:isolation & purification*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013386:Succinates / Q000737:chemistry; D013388:Succinimides / Q000737:chemistry; D053719:Tandem Mass Spectrometry / Q000379:methods
D003432:Cross-Linking Reagents; D010455:Peptides; D013386:Succinates; D013388:Succinimides; D012710:Serum Albumin, Bovine; D005982:Glutathione Transferase; C001426:N-hydroxysuccinimide