Mass spectrometric identification of formaldehyde-induced peptide modifications under in vivo protein cross-linking conditions.
Formaldehyde cross-linking of proteins is emerging as a novel approach to study protein-protein interactions in living cells. It has been shown to be compatible with standard techniques used in functional proteomics such as affinity-based protein enrichment, enzymatic digestion, and mass spectrometric protein identification. So far, the lack of knowledge on formaldehyde-induced protein modifications and suitable mass spectrometric methods for their targeted detection has impeded the identification of the different types of cross-linked peptides in these samples. In particular, it has remained unclear whether in vitro studies that identified a multitude of amino acid residues reacting with formaldehyde over the course of several days are suitable substitutes for the much shorter reaction times of 10-20 min used in cross-linking experiments in living cells. The current study on model peptides identifies amino-termini as well as lysine, tryptophan, and cysteine side chains, i.e. a small subset of those modified after several days, as the major reactive sites under such conditions, and suggests relative position in the peptide sequence as well as sequence microenvironment to be important factors that govern reactivity. Using MALDI-MS, mass increases of 12 Da on amino groups and 30 Da on cysteines were detected as the major reaction products, while peptide fragment ion analysis by tandem mass spectrometry was used to localize the actual modification sites on a peptide. Non-specific cross-linking was absent, and could only be detected with low yield at elevated peptide concentrations. The detailed knowledge on the constraints and products of the formaldehyde reaction with peptides after short incubation times presented in this study is expected to facilitate the targeted mass spectrometric analysis of proteins after in vivo formaldehyde cross-linking.
Toews
Judy
J
The Biomedical Research Centre, University of British Columbia, Vancouver, B.C., Canada.
Rogalski
Jason C
JC
Clark
Thomas J
TJ
Kast
Juergen
J
D000595:Amino Acid Sequence; D003432:Cross-Linking Reagents / Q000737:chemistry* / Q000494:pharmacology*; D005557:Formaldehyde / Q000737:chemistry* / Q000494:pharmacology*; D007185:Incubators; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010455:Peptides / Q000737:chemistry*; D011485:Protein Binding; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013997:Time Factors
D003432:Cross-Linking Reagents; D010455:Peptides; D005557:Formaldehyde