+ Site Statistics
+ Search Articles
+ PDF Full Text Service
How our service works
Request PDF Full Text
+ Follow Us
Follow on Facebook
Follow on Twitter
Follow on LinkedIn
+ Subscribe to Site Feeds
Most Shared
PDF Full Text
+ Translate
+ Recently Requested

"Out-gel" tryptic digestion procedure for chemical cross-linking studies with mass spectrometric detection

"Out-gel" tryptic digestion procedure for chemical cross-linking studies with mass spectrometric detection

Journal of Proteome Research 13(2): 527-535

SDS-PAGE is one of the most powerful protein separation techniques, and in-gel digestion is the leading method for converting proteins separated by SDS-PAGE into peptides suitable for mass spectrometry-based proteomic studies. In in-gel digestion, proteins are digested within the gel matrix, and the resulting peptides are extracted into an appropriate buffer. Transfer of the digested peptides to the liquid phase for subsequent mass spectrometric analysis, however, may be hampered by peptide-specific characteristics, including size, shape, poor solubility, adsorption to the polyacrylamide, and-in the case of cross-linking applications-by the branched structure of the peptides produced. This can be a limitation in cross-linking studies where efficient recoveries of the cross-linked peptides are critical. To overcome this limitation, we have developed a modification to the standard in-gel digestion procedure for SDS-PAGE-separated cross-linked proteins, based on older passive diffusion methods. By omitting the gel staining and gel fixation steps, intact proteins or cross-linked protein complexes can move through the gel and into the buffer solution. Digestion of the entire protein in the buffer outside the gel increases the probability that most of the proteolytic peptides produced will be present in the final digest solution. The resulting peptide mixture is then freed of SDS and concentrated using SCX (strong cation exchange) zip-tips and analyzed by mass spectrometry. For standard protein identification studies and the recovery of noncross-linked peptides, the in-gel procedure outperformed the out-gel procedure, but for cross-linking studies with enrichable cross-linkers (such as CBDPS), the standard out-gel procedure allowed the recoveries of cross-links not recovered via the in-gel method. Labeling experiments showed that, with an enrichable cross-linker, 93% of the cross-links showed better or equal recoveries with the out-gel procedure, as compared to the in-gel procedure. It should be noted that this method is not designed to replace in-gel digestion for most proteomics applications. However, by using the out-gel method, we were able to detect twice as many interprotein CBDPS cross-links from the histone H2A/H2B complex as were found in the in-gel digested sample.

Please choose payment method:

(PDF emailed within 0-6 h: $19.90)

Accession: 051007275

Download citation: RISBibTeXText

PMID: 24354799

DOI: 10.1021/pr400710q

Related references

Accelerated tryptic digestion for the analysis of biopharmaceutical monoclonal antibodies in plasma by liquid chromatography with tandem mass spectrometric detection. Journal of Chromatography. a 1217(1): 57-64, 2010

Chemical cross-linking and mass spectrometric identification of sites of interaction for UreD, UreF, and urease. Journal of Biological Chemistry 279(15): 15305-15313, 2004

Bioconjugation of ribonuclease A: A detailed chromatographic and mass spectrometric analysis of chemical modification by a cross-linking reagent. Bioconjugate Chemistry 11(2): 182-194, March-April, 2000

Chemical cross-linking with thiol-cleavable reagents combined with differential mass spectrometric peptide mapping--a novel approach to assess intermolecular protein contacts. Protein Science 9(8): 1503-1518, 2000

A comparison of different biotinylation reagents, tryptic digestion procedures, and mass spectrometric techniques for 2-D peptide mapping of membrane proteins. Proteomics 5(12): 3035-3039, 2005

Use of activated graphitized carbon chips for liquid chromatography/mass spectrometric and tandem mass spectrometric analysis of tryptic glycopeptides. Rapid Communications in Mass Spectrometry 23(4): 495-505, 2009

One-step procedure for peptide extraction from in-gel digestion sample for mass spectrometric analysis. Analytical Chemistry 80(24): 9797-9805, 2008

Cyanogen bromide peptides of the fibrillar collagens I, III, and V and their mass spectrometric characterization: detection of linear peptides, peptide glycosylation, and cross-linking peptides involved in formation of homo- and heterotypic fibrils. Journal of Proteome Research 6(11): 4269-4289, 2007

New designer drug 4-iodo-2,5-dimethoxy-beta-phenethylamine (2C-I): studies on its metabolism and toxicological detection in rat urine using gas chromatographic/mass spectrometric and capillary electrophoretic/mass spectrometric techniques. Journal of Mass Spectrometry 41(7): 872-886, 2006

A new approach to protein structural studies using chemical cross-linking and top-down fourier transform mass spectrometry. Biophysical Journal 84(2 Part 2): 356a, 2003

A top-down approach to protein structure studies using chemical cross-linking and Fourier transform mass spectrometry. European Journal of Mass Spectrometry 9(6): 623-631, 2003

A top down approach to protein structural studies using chemical cross-linking and Fourier transform mass spectrometry. Rapid Communications in Mass Spectrometry 17(2): 155-162, 2003

Mass spectrometric analysis of cross-linking sites for the structure of proteins and protein complexes. Molecular Biosystems 4(8): 816-823, 2008

Application of a fast sorting algorithm to the assignment of mass spectrometric cross-linking data. Proteomics 14(17-18): 1987-1989, 2014

Isotope-tagged cross-linking reagents. A new tool in mass spectrometric protein interaction analysis. Analytical Chemistry 73(9): 1927-1934, 2001