An accurate proteomic quantification method: fluorescence labeling absolute quantification (FLAQ) using multidimensional liquid chromatography and tandem mass spectrometry.
A facile proteomic quantification method, fluorescent labeling absolute quantification (FLAQ), was developed. Instead of using MS for quantification, the FLAQ method is a chromatography-based quantification in combination with MS for identification. Multidimensional liquid chromatography (MDLC) with laser-induced fluorescence (LIF) detection with high accuracy and tandem MS system were employed for FLAQ. Several requirements should be met for fluorescent labeling in MS identification: Labeling completeness, minimum side-reactions, simple MS spectra, and no extra tandem MS fragmentations for structure elucidations. A fluorescence dye, 5-iodoacetamidofluorescein, was finally chosen to label proteins on all cysteine residues. The fluorescent dye was compatible with the process of the trypsin digestion and MALDI MS identification. Quantitative labeling was achieved with optimization of reacting conditions. A synthesized peptide and model proteins, BSA (35 cysteines), OVA (five cysteines), were used for verifying the completeness of labeling. Proteins were separated through MDLC and quantified based on fluorescent intensities, followed by MS identification. High accuracy (RSD% < 1.58) and wide linearity of quantification (1-10(5) ) were achieved by LIF detection. The limit of quantitation for the model protein was as low as 0.34 amol. Parts of proteins in human liver proteome were quantified and demonstrated using FLAQ.
Department of Chemistry & Institutes of Biomedical Sciences, Fudan University, Shanghai, China.
Junyan
J
Liu
Yang
Y
Liu
Mingxia
M
Gao
Xiangmin
X
Zhang
D000818:Animals; D002417:Cattle; D002852:Chromatography, Ion Exchange / Q000379:methods*; D003545:Cysteine / Q000032:analysis / Q000737:chemistry; D005452:Fluoresceins / Q000032:analysis / Q000737:chemistry; D005456:Fluorescent Dyes / Q000032:analysis / Q000737:chemistry; D006801:Humans; D016014:Linear Models; D008099:Liver / Q000737:chemistry; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D055550:Protein Stability; D011506:Proteins / Q000032:analysis* / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014357:Trypsin / Q000378:metabolism
D005452:Fluoresceins; D005456:Fluorescent Dyes; D010446:Peptide Fragments; D011506:Proteins; C015296:5-iodoacetamidofluorescein; D014357:Trypsin; D003545:Cysteine