By Nguyet Thuy Tran, Myriam Taverna
This publication offers a accomplished survey of contemporary advancements and purposes of excessive functionality capillary electrophoresis within the box of protein and peptide research with a unique specialize in the research of intact proteins. With useful element, the contents hide diversified modes of capillary electrophoresis (CE) beneficial for protein and peptide research, CZE, CIEF, ACE, CGE, and sorts of program corresponding to the standard keep watch over of healing proteins and monoclonal antibodies, medical analyses of chemokines in tissues, qualitative and quantitative research of vaccine proteins, and resolution of binding constants in complexes concerning peptides or proteins. Written for the hugely profitable Methods in Molecular Biology sequence, chapters contain introductions to their respective issues, lists of the mandatory fabrics and reagents, step by step, simply reproducible laboratory protocols, and pointers on troubleshooting and heading off recognized pitfalls.
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Extra info for Capillary Electrophoresis of Proteins and Peptides: Methods and Protocols
The tip of the stainless steel tube serves also as electrospray needle. Typical voltages between the sprayer and the CE-MS of Proteins a 27 stainless-steel capillary nebulizing gas CE capillary MS analyzer sheath liquid electrical contact b stainless-steel capillary polyimide fused silica static conductive liquid CE capillary MS analyzer electrical contact porous tip (length, ~40 mm; protrusion, 5 mm; wall thickness, 5 µm) Fig. 1 Schematic representation of a sheath-liquid (a) and a sheathless (b) CE-MS interface entrance of the mass spectrometer are in the range of 2–5 kV.
Proteins are generally determined in positive ion mode as they are effectively protonated to form positively charged ions during ESI. A third coaxial tube delivers a gas flow that aids nebulization of the CE effluent into the ion source. The nebulizer gas can produce a small suction of the CE effluent. This effect could cause band broadening and decrease in separation efficiency and resolution and, therefore, should be evaluated. The sheath-liquid composition is essential [7, 8], because it importantly determines the protein signal intensity as well as the shape and position of the protein's charge state distribution.
3. 2. Install the nanospray end plate and gas diverter to allow nanoESI and the XYZ-stage for the porous-tip capillaries. 4. Place the capillary in the XYZ-stage and position the spray tip at a distance of 3 mm with respect to the MS source ( see Note 15 ). 5. Change the MS configuration to nanoESI. 6. Select the MS method and set the drying gas at 3 L/min N2 and 180 °C and the scan range 300–3000 m/z (see Note 16). CE-MS of Proteins b migr. 0 6 7 8 9 time (min) 10 2x 11 Fig. 0). (b) Migration time, molecular mass, and glycan composition for rhIFN-β glycoforms observed.
Capillary Electrophoresis of Proteins and Peptides: Methods and Protocols by Nguyet Thuy Tran, Myriam Taverna