We show that this concept allows for facile identification of labeled cellular domains, simultaneously with mapping of the macromolecules using spontaneous Raman technique.
The H2B-GFP fusion protein makes the fluorescent label very stable and the nuclear localization also facilitates easier identification of label-retaining cells in histological sections.
In all cases, including a dibromide signature in the covalently bound tag could improve detection and identification of labeled peptides.
These properties, linked with the potential for affinity purification and MS-based identification of labeled enzymes, markedly enhance the utility of the approach.
Synthetic and biochemical methods for the generation and characterization of the metabolic chemical reporter, additional data figures, and proteomic identification of labeled proteins.
Its normalization feature enables the easy identification of labels that are correlated with the primary data, while at the same time a p-value measures the level of significance of the detected correlations.
The mass spectrometer was calibrated on selected fragments from the MS/MS spectrum of Glu-Fibrinopeptide B. MS/MS spectra were submitted to Mascot for identification of labeled peptides and amino acids (13).
This was even more apparent when 5-HT2C mRNA was detected using MS-TSA-FISH, where the signal-to-noise ratio was too low to allow unequivocal identification of labeled neurons (Fig. 2e).
Black-and-white dark-field images were converted to reverse-red grains and superimposed upon standard light microscope images using Adobe Photoshop (Adobe Systems Inc., San Jose, CA), allowing for easy identification of labeled structures.
Because the SEQUEST algorithm allows some deviation between the theoretical mass of a peptide and the mass observed by the mass spectrometer, assuming an additional mass of 0.95 Daltons/nitrogen atom would not necessarily preclude identification of labeled proteins with similar levels of N incorporation (e.g., 92% labeled peptides may still be identified).
