Strong Cation Exchange Monolith Research Articles

Silica-supported polymeric monolithic column with a mixed mode of hydrophilic and strong cation-exchange interactions for microcolumn liquid chromatography.

A novel sulfonic acid group containing hydrophilic strong cation-exchange monolith was prepared by in situ coating 5 μm bare silica particles with the copolymers of glycidyl methacrylate and pentaerythritol triacrylate and further sulfonating the prepared polymer matrix with Na2 SO3 inside a 150 μm id capillary. The preparation conditions were investigated, and the method was described in detail. The prepared column was characterized by comparing with its counterparts reported previously in terms of matrix morphology, preparation reproducibility, permeability, swelling-shrinking behavior, mechanical stability, hydrophilicity, binding capacity, and column efficiency. The swelling-shrinking behavior of the present column in solvents of different polarities was negligible, the hydrophobicity could be suppressed at the acetonitrile concentrations higher than 40% v/v, and the binding capacities were 256 μequiv/mL and 20.1 mg/mL for Cu2+ and lysozyme, respectively. The minimum theoretical plate heights were 8, 10, and 13 μm, and the values of the C term in van Deemter equation were 9, 12, and 35 ms for the test analytes of Na+ , thiourea, and cytidine 5'-monophosphate, respectively. This column exhibited an excellent performance in the separations of monovalent inorganic cations, uncharged polar, and charged polar compounds.

Comprehensive analysis of host cell impurities in monoclonal antibodies with improved sensitivity by capillary zone electrophoresis mass spectrometry.

Four methods were compared for analysis of host-cell protein (HCP) impurities in a recombinant mAb. First, CZE-MS/MS was used to analyze the digest of an HCP sample following extraction of the mAb with proteins A and L affinity columns; 220 protein groups and 976 peptides were identified from the depleted HCP digest. Second, a nanoACQUITY UltraPerformance LCH system was also used to analyze the depleted HCP digest; 34 protein groups and 53 peptides from 50 ng of the depleted HCP digest and 290 protein groups and 1011 peptides were identified from 1 μg of the depleted HCP digest. Third, 185 protein groups and 709 peptides were identified by CZE-MS/MS from the HCP digest without depletion. Fourth, a strong cation exchange SPE was coupled to CZE-ESI-MS/MS using online pH gradient elution for analysis of the HCP digest without depletion. A series of five pH bumps were applied to elute peptides from the strong cation exchange monolith followed by analysis using CZE coupled to a Q Exactive HF mass spectrometer; 230 protein groups and 796 peptides were identified from the HCP digest without depletion.

Nearly 1000 Protein Identifications from 50 ng of Xenopus laevis Zygote Homogenate Using Online Sample Preparation on a Strong Cation Exchange Monolith Based Microreactor Coupled with Capillary Zone Electrophoresis.

A sulfonate-silica hybrid strong cation exchange monolith microreactor was synthesized and coupled to a linear polyacrylamide coated capillary for online sample preparation and capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) bottom-up proteomic analysis. The protein sample was loaded onto the microreactor in an acidic buffer. After online reduction, alkylation, and digestion with trypsin, the digests were eluted with 200 mM ammonium bicarbonate at pH 8.2 for CZE-MS/MS analysis using 1 M acetic acid as the background electrolyte. This combination of basic elution and acidic background electrolytes results in both sample stacking and formation of a dynamic pH junction. 369 protein groups and 1274 peptides were identified from 50 ng of Xenopus laevis zygote homogenate, which is comparable with an offline sample preparation method, but the time required for sample preparation was decreased from over 24 h to less than 40 min. Dramatically improved performance was produced by coupling the reactor to a longer separation capillary (∼100 cm) and a Q Exactive HF mass spectrometer. 975 protein groups and 3749 peptides were identified from 50 ng of Xenopus protein using the online sample preparation method.

Detachable strong cation exchange monolith, integrated with capillary zone electrophoresis and coupled with pH gradient elution, produces improved sensitivity and numbers of peptide identifications during bottom-up analysis of complex proteomes.

A detachable sulfonate-silica hybrid strong cation-exchange monolith was synthesized in a fused silica capillary, and used for solid phase extraction with online pH gradient elution during capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) proteomic analysis. Tryptic digests were prepared in 50 mM formic acid and loaded onto the strong cation-exchange monolith. Fractions were eluted using a series of buffers with lower concentration but higher pH values than the 50 mM formic acid background electrolyte. This combination of elution and background electrolytes results in both sample stacking and formation of a dynamic pH junction and allows use of relatively large elution buffer volumes while maintaining reasonable peak efficiency and resolution. A series of five pH bumps were applied to elute E. coli tryptic peptides from the monolith, followed by analysis using CZE coupled to an LTQ-Orbitrap Velos mass spectrometer; 799 protein groups and 3381 peptides were identified from 50 ng of the digest in a 2.5 h analysis, which approaches the identification rate for this organism that was obtained with an Orbitrap Fusion. We attribute the improved numbers of peptide and protein identifications to the efficient fractionation by the online pH gradient elution, which decreased the complexity of the sample in each elution step and improved the signal intensity of low abundance peptides. We also performed a comparative analysis using a nanoACQUITY UltraPerformance LCH system. Similar numbers of protein and peptide identifications were produced by the two methods. Protein identifications showed significant overlap between the two methods, whereas peptide identifications were complementary.

A simple and efficient method for preparation of a strong cation exchange monolith and its application to separation of HSA

A strong cation exchange monolithic column was prepared by a single step in situ radical polymerization without further chemical modification for high performance liquid chromatography (HPLC). The novel monolithic column used vinyl ester resin as the monomer, ethylene glycol dimethacrylate (EDMA) as the cross linking agent, 2,2′-azobisisobutyronitrile (AIBN) as the initiating agent, NaHSO3 both as inorganic adjunct and coadunate initiator (L. G. Bai, H. Y. Liu, Y. K. Liu, J. Chromatogr. A, 2010, 1218, 100-106, and F. A. Zhang, C. L. Yu, X. H. Zhu, C. Wei, Polym. Adv. Technol., 2006, 17, 608–611). Moreover, NaHSO3 can supply sulfonic acid groups as cation exchangers for ion-exchange monolithic column. Polymerization conditions and separation conditions were optimized, and the structure properties of the monoliths were also investigated. In addition, the chromatographic performance of the monolith was evaluated through separating human serum albumin (HSA) from human plasma in conjunction with HPLC and the expected results were acquired. The effects of buffer concentration and pH on the separation of HSA were investigated. Furthermore, the monolith was used to separate the mixture of protein (hemoglobin and HSA) with good resolution.

Rare Cell Proteomic Reactor Applied to Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)-based Quantitative Proteomics Study of Human Embryonic Stem Cell Differentiation

The molecular basis governing the differentiation of human embryonic stem cells (hESCs) remains largely unknown. Systems-level analysis by proteomics provides a unique approach to tackle this question. However, the requirement of a large number of cells for proteomics analysis (i.e. 10(6)-10(7) cells) makes this assay challenging, especially for the study of rare events during hESCs lineage specification. Here, a fully integrated proteomics sample processing and analysis platform, termed rare cell proteomic reactor (RCPR), was developed for large scale quantitative proteomics analysis of hESCs with ∼50,000 cells. hESCs were completely extracted by a defined lysis buffer, and all of the proteomics sample processing procedures, including protein preconcentration, reduction, alkylation, and digestion, were integrated into one single capillary column with a strong cation exchange monolith matrix. Furthermore, on-line two-dimensional LC-MS/MS analysis was performed directly using RCPR as the first dimension strong cation exchange column. 2,281 unique proteins were identified on this system using only 50,000 hESCs. For stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative study, a ready-to-use and chemically defined medium and an in situ differentiation procedure were developed for complete SILAC labeling of hESCs with well characterized self-renewal and differentiation properties. Mesoderm-enriched differentiation was studied by RCPR using 50,000 hESCs, and 1,086 proteins were quantified with a minimum of two peptides per protein. Of these, 56 proteins exhibited significant changes during mesoderm-enriched differentiation, and eight proteins were demonstrated for the first time to be overexpressed during early mesoderm development. This work provides a new platform for the study of rare cells and in particular for further elucidating proteins that govern the mesoderm lineage specification of human pluripotent stem cells.

A weak cation-exchange monolith as stationary phase for the separation of peptide diastereomers by CEC

A CEC weak cation-exchange monolith has been prepared by in situ polymerization of acrylamide, methylenebisacrylamide and 4-acrylamidobutyric acid in a decanol-dimethylsulfoxide mixture as porogen. The columns were evaluated by SEM and characterized with regard to the separation of diastereomers and α/β-isomers of aspartyl peptides. Column preparation was reproducible as evidenced by comparison of the analyte retention times of several columns prepared simultaneously. Analyte separation was achieved using mobile phases consisting of acidic phosphate buffer and ACN. Under these conditions the peptides migrated due to their electrophoretic mobility but the EOF also contributed as driving force as a function of the pH of the mobile phase due to increasing dissociation of the carboxyl groups of the polymer. Raising the pH of the mobile phase also resulted in deprotonation of the peptides reducing analyte mobility. Due to these mechanisms each pair of diastereomeric peptides displayed the highest resolution at a different pH of the buffer component of the mobile phase. Comparing the weak-cation exchange monolith to an RP monolith and a strong cation-exchange monolith different elution order of some peptide diastereomers was observed, clearly illustrating that interactions with the stationary phase contribute to the CEC separations.

Capillary Trap Column with Strong Cation-Exchange Monolith for Automated Shotgun Proteome Analysis

A 150 microm internal diameter capillary monolithic column with a strong cation-exchange stationary phase was prepared by direct in situ polymerization of ethylene glycol methacrylate phosphate and bisacrylamide in a trinary porogenic solvent consisting dimethylsulfoxide, dodecanol, and N,N'-dimethylformamide. This phosphate monolithic column exhibits higher dynamic binding capacity, faster kinetic adsorption of peptides, and more than 10 times higher permeability than the column packed with commercially available strong cation-exchange particles. It was applied as a trap column in a nanoflow liquid chromatography-tandem mass spectrometry system for automated sample injection and online multidimensional separation. It was observed that the sample could be loaded at a flow rate as high as 40 microL/min with a back pressure of approximately 1300 psi and without compromising the separation efficiency. Because of its good orthogonality to the reversed phase separation mechanism, the phosphate monolithic trap column was coupled with a reversed-phase column for online multidimensional separation of 19 microg of the tryptic digest of yeast proteins. A total of 1522 distinct proteins were identified from 5608 unique peptides (total of 54,780 peptides) at the false positive rate only 0.46%.