N-terminal tagging strategy for de novo sequencing of short peptides by ESI-MS/MS and MALDI-MS/MS.
The major portion of skin secretory peptidome of the European Tree frog Hyla arborea consists of short peptides from tryptophyllin family. It is known that b-ions of these peptides undergo head-to-tail cyclization, forming a ring that can open, resulting in several linear forms. As a result, the spectrum contains multiple ion series, thus complicating de novo sequencing. This was observed in the Q-TOF spectrum of one of the tryptophyllins isolated from Hyla arborea; the sequence FLPFFP-NH(2) was established by Edman degradation and counter-synthesis. Though no rearrangements were observed in FTICR-MS and MALDI-TOF/TOF spectra, both of them were not suitable for mass-spectrometry sequencing due to the low sequence coverage. To obtain full amino acid sequence by mass spectrometry, three chemical modifications to N-terminal amino moiety were applied. They include acetylation and sulfobenzoylation of N-amino group and its transformation to 2,4,6-trimethylpyridinium by interaction with 2,4,6-trimethylpyrillium tetrafluoroborate. All three reagents block scrambling and provide spectra better than the intact peptide. Unfortunately, all of them also readily react with lysine side chain. Hence, all investigated procedures can be used to improve sequencing of short peptides, while acetylation is the recommended one. It shows excellent results, and it is plain and simple to perform. This is the procedure of choice for MS-sequencing of short peptides by manual or automatic algorithms.
Samgina
Tatiana Yu
TY
Organic Chemistry Department, Moscow State University, Moscow, Russia.
Kovalev
Sergey V
SV
Gorshkov
Vladimir A
VA
Artemenko
Konstantin A
KA
Poljakov
Nikita B
NB
Lebedev
Albert T
AT
D000595:Amino Acid Sequence; D000818:Animals; D001001:Anura / Q000378:metabolism*; D009842:Oligopeptides / Q000737:chemistry* / Q000302:isolation & purification; D020539:Sequence Analysis, Protein / Q000379:methods*; D012867:Skin / Q000737:chemistry* / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods
D009842:Oligopeptides; C048291:tryptophyllins