Real-time laser induced chemical derivatizations of peptide N-Terminus for in-situ mass spectrometric sequencing at sub-picomole and nanosecond scale.

Distinguishing b- and y-ions is essential to compute amino acid sequences from either N- or C-terminus in mass spectrometry. We described herein a solvent free and real time on-plate derivatization approach that can tag N-terminus of peptides at microliter level with p-chlorobenzaldehyde or 2-hydroxy-5-methylisophthalaldehyde for matrix assisted laser desorption ionization mass spectrometry (MALDI MS). Less than 1 μL of sample solutions can be directly mixed with equal volumes of p-chlorobenzaldehyde or 2-hydroxy-5-methylisophthalaldehyde and α-cyano-4-hydroxycinnamic acid (CHCA), a matrix compound to co-crystalize with analytes for efficient absorption of laser energy and peptide ionization. When the mixture spotted on the sample plate is irradiated with the 3rd harmonic (355 nm) of Nd3+:YAG laser pulses (3 ns width), N-terminal amine groups of peptides instantly react with carbonyl groups of chlorobenzaldehyde or 2-hydroxy-5-methylisophthalaldehyde. Resultant peptides carrying with on-plate formed azomethine group (-CN-) are simultaneously protonated and isolated as precursor ions for subsequent collision-activated dissociation. The mass shift with unique Cl isotopic signature unambiguously distinguishes b ions from y ions and other ions. This method does not need extensive sample preparation and is useful for those samples with limited quantities down to sub-picomole level in sub-microliter volumes. The efficiency was demonstrated with synthetic peptides and tryptic peptides of model proteins. It was found that 2-hydroxy-5-methylisophthalaldehyde provides improved yield for peptides containing lysine residues. Unknown proteins of human saliva and bovine milk as well as phosphopeptides have been identified.