Detection of oligosaccharides labeled with cyanine dyes using matrix-assisted laser desorption/ionization mass spectrometry.
The sensitivity of oligosaccharides in mass spectrometry lags far behind that of peptides. This is a critical factor in realizing the high-throughput analysis of posttranslational modifications in proteomics. We here described that hydrazide derivatives of cyanine dyes (Cy3, Cy5) with a positive charge made excellent labeling reagents for the detection of oligosaccharides by matrix-assisted laser desorption/ionization mass spectrometry. Cy3-labeled standard N-glycan could be detected at 200 amol on the MALDI target plate in reflectron mode without any purification procedures after the labeling reaction, which may meet the level of sensitivity required in proteome research. Despite the general recognition that the production of signals of oligosaccharides under MALDI conditions would be highly dependent on the matrix, most of the known N-glycans from chicken ovalbumin could be detected upon Cye derivatization nearly independent of the kind of matrix tested (e.g., nor-harman, 2,5-dihydroxybenzoic acid and alpha-cyano-4-hydroxycinnamic acid) without spoiling the signal strength. Postsource decay afforded simple spectra mainly consisting of Y-type fragment ions, thus simplifying the sequence analysis. In-source decay afforded a similar fragmentation pattern only when acidic matrixes were used. In addition, this derivatization technique was successfully applied to the profiling of N-glycans of gel-separated glycoproteins.
Kameyama
Akihiko
A
Department of Research and Development, Amersham Biosciences K.K., 3-25-1 Hyakunincho, Shinjuku-ku Tokyo 169-0073, Japan. aki-kameyama@aist.go.jp
Kaneda
Yuko
Y
Yamanaka
Hidenori
H
Yoshimine
Hiroshi
H
Narimatsu
Hisashi
H
Shinohara
Yasuro
Y
D002232:Carbocyanines; D002240:Carbohydrate Sequence; D006023:Glycoproteins / Q000737:chemistry* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000032:analysis*; D011134:Polysaccharides / Q000032:analysis; D040901:Proteomics; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
D002232:Carbocyanines; D006023:Glycoproteins; D009844:Oligosaccharides; D011134:Polysaccharides; C085321:cyanine dye 5