Top-down FTICR MS for the identification of fluorescent labeling efficiency and specificity of the Cu-protein azurin.
Fluorescent protein labeling has been an indispensable tool in many applications of biochemical, biophysical, and cell biological research. Although detailed information about the labeling stoichiometry and exact location of the label is often not necessary, for other purposes, this information is crucial. We have studied the potential of top-down electrospray ionization (ESI)-15T Fourier transform ion cyclotron resonance (FTICR) mass spectrometry to study the degree and positioning of fluorescent labeling. For this purpose, we have labeled the Cu-protein azurin with the fluorescent label ATTO 655-N-hydroxysuccinimide(NHS)-ester and fractionated the sample using anion exchange chromatography. Subsequently, individual fractions were analyzed by ESI-15T FTICR to determine the labeling stoichiometry, followed by top-down MS fragmentation, to locate the position of the label. Results showed that, upon labeling with ATTO 655-NHS, multiple different species of either singly or doubly labeled azurin were formed. Top-down fragmentation of different species, either with or without the copper, resulted in a sequence coverage of approximately 50%. Different primary amine groups were found to be (potential) labeling sites, and Lys-122 was identified as the major labeling attachment site. In conclusion, we have demonstrated that anion exchange chromatography in combination with ultrahigh resolution 15T ESI-FTICR top-down mass spectrometry is a valuable tool for measuring fluorescent labeling efficiency and specificity.
Biomolecular Mass Spectrometry Unit, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden, The Netherlands.
Simone
S
Nicolardi
Alessio
A
Andreoni
Leandro C
LC
Tabares
Yuri E M
YE
van der Burgt
Gerard W
GW
Canters
André M
AM
Deelder
Paul J
PJ
Hensbergen
D001400:Azurin / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D002852:Chromatography, Ion Exchange; D003300:Copper / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry*; D005583:Fourier Analysis; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization
D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D001400:Azurin; D003300:Copper