Continuous affinity-gradient nano-stationary phase served as a column for reversed-phase electrochromatography and matrix carrier in time-of-flight mass spectrometry for protein analysis.
This study developed an affinity-gradient nano-stationary phase (AG-NSP) for protein analysis using nanofluidic capillary electrochromatography (nano-CEC) conjugated with matrix assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS). The AG-NSP can be used for protein pre-separation in nano-CEC and as a matrix carrier for protein analysis in MALDI-TOF-MS. A hydrophobicity gradient in AG-NSP was photochemically formed by grafting 4-azidoaniline hydrochloride on vertically arrayed multi-wall carbon nanotubes (MWCNTs) through gray-level exposure to UV light. The reversed-phase gradient stationary phase in AG-NSP was tailored according to the properties of the mobile phase gradient in capillary electrochromatography. As a result, the operation of the system is easily automated using a single buffer solution without the need for multiple solvents for elution. The use of nano-CEC with AG-NSP demonstrated excellent separation efficiency and high resolution for various types of DNA/protein/peptide. MALDI-TOF-MS analysis was then performed directly on the separated proteins and peptides on the chip. The proposed system was then used for the detection of three types of proteins with different molecular weights and PI values, including Cytochrome c (12,360, pI = 10), Lysozyme (14,300, pI = 11), and BSA (86,000, pI = 5)), and digested IgG fragments. The proposed system provided resolution of 1000 Da for the proteins in this study and the separation of digested IgG fragments at a low concentration of 1.2 pmol μL(-1).
Department of Engineering and System Science, National Tsing Hua University, 101, Section 2 Kuang Fu Road, Hsinchu 30013, Taiwan, ROC.
Jen-Kuei
JK
Wu
National Health Research Institutes, 35 Keyan Road, Zhunan, Miaoli County 35053, Taiwan, ROC.
Chung-Shi
CS
Yang
Department of Engineering and System Science, National Tsing Hua University, 101, Section 2 Kuang Fu Road, Hsinchu 30013, Taiwan, ROC.
Yi-Shiuan
YS
Wu
Department of Engineering and System Science, National Tsing Hua University, 101, Section 2 Kuang Fu Road, Hsinchu 30013, Taiwan, ROC.
Pen-Cheng
PC
Wang
Department of Engineering and System Science, National Tsing Hua University, 101, Section 2 Kuang Fu Road, Hsinchu 30013, Taiwan, ROC; Division of Mechanics, Research Center for Applied Sciences, Academia Sinica, Taipei, Taiwan, ROC. Electronic address: fangang@ess.nthu.edu.tw.
Fan-Gang
FG
Tseng
D053801:Capillary Electrochromatography; D002851:Chromatography, High Pressure Liquid; D056148:Chromatography, Reverse-Phase; D045304:Cytochromes c / Q000032:analysis; D007074:Immunoglobulin G / Q000032:analysis / Q000378:metabolism; D009113:Muramidase / Q000032:analysis; D036103:Nanotechnology; D011506:Proteins / Q000032:analysis*; D012710:Serum Albumin, Bovine / Q000032:analysis; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000295:instrumentation / Q000379:methods*
D007074:Immunoglobulin G; D011506:Proteins; D012710:Serum Albumin, Bovine; D045304:Cytochromes c; D009113:Muramidase
Affinity-gradient nano-stationary phase; MWCNTs nanostructured chromatographic supports; Matrix carrier; Nanofluidic capillary electrochromatography