ABSTRACT
A sheathless interface making use of a porous tip has been used for coupling capillary electrophoresis and electrospray ionization mass spectrometry. First, effective flow rates using the interface have been characterized. It was found that the interface is capable of generating a stable spray with flow rates ranging from below 10 nL/min to >340 nL/min, enabling its use in either the mass or concentration-sensitive region of the electrospray process. Subsequently, by analyzing peptide mixtures of increasing complexity, we have demonstrated that this platform provides exquisite sensitivity enabling the detection of very low amounts of materials with very high resolving power. Transient isotachophoresis (t-ITP) can also be integrated with this setup to increase the mass loading of the system while maintaining peak efficiency and resolution. Concentration limits of detection in the subnanomolar or nanomolar range can be achieved with or without t-ITP, respectively. The application of a vacuum at the inlet of the separation capillary further allowed the peak capacity of the system to be improved while also enhancing its efficiency. As a final step in this study, it was demonstrated that the intrinsic properties of the interface allows the use of coated noncharged surfaces so that very high peak capacities can be achieved.
Subject(s)
Electrophoresis, Capillary/methods , Isotachophoresis/methods , Spectrometry, Mass, Electrospray Ionization/methods , Animals , Cattle , Electricity , Hydrofluoric Acid/chemistry , Kinetics , Peptides/analysis , Peptides/isolation & purification , PorosityABSTRACT
Separation of complex protein mixtures that have a wide dynamic range of concentration, such as plasma or serum, is a challenge for proteomic analysis. Sample preparation to remove high-abundant proteins is essential for proteomics analysis. Immunoglobulin yolk (IgY) antibodies have unique and advantageous features that enable specific protein removal to aid in the detection of low-abundant proteins and biomarker discovery. This report describes the efficiency and effectiveness of IgY microbeads in separating 12 abundant proteins from plasma with an immunoaffinity spin column or LC column. The protein separation and sample preparation process was monitored via SDS-PAGE, 2-DE, LC-MS/MS, or clinical protein assays. The data demonstrate the high specificity of the protein separation, with removal of 95-99.5% of the abundant proteins. IgY microbeads against human proteins can also selectively remove orthologous proteins of other mammals such as mouse, rat, etc. Besides the specificity and reproducibility of the IgY microbeads, the report discusses the factors that may cause potential variations in protein separation such as protein-protein interactions (known as "Interactome"), binding and washing conditions of immunoaffinity reagents, etc. A novel concept of Seppromics is introduced to address methodologies and science of protein separation in a context of proteomics.
Subject(s)
Blood Proteins/chemistry , Blood Proteins/isolation & purification , Immunoglobulins/chemistry , Microspheres , Proteomics/methods , Albumins/chemistry , Animals , Biomarkers/chemistry , Chromatography, Liquid , Edetic Acid/chemistry , Electrophoresis, Gel, Two-Dimensional , Electrophoresis, Polyacrylamide Gel , Humans , Mass Spectrometry , MiceABSTRACT
A major challenge in protein target discovery and validation is how to specifically dissect complex protein mixtures and measure trace targets. Immunoaffinity-based protein capture, separation and detection have proven to be one of the most effective approaches. Avian IgY antibody microbeads (Seppro™), representing a type of novel and specific protein sorbent, have several distinct advantages over IgG. Their utility and applications are compared with those of IgG and other affinity reagents.:
