Performance comparison of three trypsin columns used in liquid chromatography

Trypsin is the most widely used enzyme in proteomic research due to its high specificity. Although the in-solution digestion is predominantly used, it has several drawbacks, such as long digestion times, autolysis, and intolerance to high temperatures or organic solvents. To overcome these shortcomings trypsin was covalently immobilized on solid support and tested for its proteolytic activity. Trypsin was immobilized on bridge-ethyl hybrid silica sorbent with 300 Å pores, packed in 2.1 × 30 mm column and compared with Perfinity and Poroszyme trypsin columns. Catalytic efficiency of enzymatic reactors was tested using Nα-Benzoyl-L-arginine 4-nitroanilide hydrochloride as a substrate. The impact of buffer pH, mobile phase flow rate, and temperature on enzymatic activity was investigated. Digestion speed generally increased with the temperature from 20 to 37 °C. Digestion speed also increased with pH from 7.0 to 9.0; the activity of prototype enzyme reactor was highest at pH 9.0, when it activity exceeded both commercial reactors. Preliminary data for fast protein digestion are presented

Strukturní stabilita oceli Cr15Ni6Mi

Import 20/04/2006Prezenční výpůjčkaVŠB - Technická univerzita Ostrava. Fakulta metalurgie a materiálového inženýrství. Katedra (636) materiálového inženýrstv

Peak capacity in gradient reversed-phase liquid chromatography of biopolymers Theoretical and practical implications for the separation of oligonucleotides

Abstract Reversed-phase ultra-performance liquid chromatography was used for biopolymer separations in isocratic and gradient mode. The gradient elution mode was employed to estimate the optimal mobile phase flow rate to obtain the best column efficiency and the peak capacity for three classes of analytes: peptides, oligonucleotides and proteins. The results indicate that the flow rate of the Van Deemter optimum for 2.1 mm I.D. columns packed with a porous 1.7 m C 18 sorbent is below 0.2 mL/min for our analytes. However, the maximum peak capacity is achieved at flow rates between 0.15 and 1.0 mL/min, depending on the molecular weight of the analyte. The isocratic separation mode was utilized to measure the dependence of the retention factor on the mobile phase composition. Constants derived from isocratic experiments were utilized in a mathematical model based on gradient theory. Column peak capacity was predicted as a function of flow rate, gradient slope and column length. Predicted peak capacity trends were compared to experimental results

Ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) and UPLC/MS E analysis of RNA oligonucleotides

Fast and efficient ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) analysis of short interfering RNA oligonucleotides was used for identity confirmation of the target sequence-related impurities. Multiple truncated oligonucleotides and metabolites were identified based on the accurate mass, and their presumed sequence was confirmed by MS/MS and MS E (alternating low and elevated collision energy scanning modes) methods. Synthetic RNA oligonucleotides need to be purified to avoid off-target silencing of undesirable genes. When intended for use as drugs, RNA oligonucleotides and their duplexes need to be well characterized to satisfy regulatory requirements and minimize possible adverse effects. Modifications of RNA molecules are often introduced in order to improve their enzymatic stability, cell membrane permeability, or increase the half-life of siRNA duplexes. However, the modifications make analysis of modified RNA molecules more challenging. 4 The ion-pairing reversed-phase (IP RP) high-performance liquid chromatographic (HPLC) method is commonly applied for the quantitation and characterization of oligonucleotides samples. 13,14 While LC/MS provides accurate mass and can be used to assign the identity of target peaks and failure sequences, in some cases the characterization of therapeutic oligonucleotides requires detailed sequence information. Sequencing of oligonucleotides is traditionally performed by selective enzymatic or chemical cleavage using the Maxam-Gilbert method or using exonucleases to create ladders that are examined by capillary electrophoresis 15 or mass spectrometry

Liquid Chromatography Methods for Analysis of mRNA Poly(A) Tail Length and Heterogeneity

Messenger RNA (mRNA) is a new class of therapeutic compounds. The current advances in mRNA technology require the development of efficient analytical methods. In this work, we describe the development of several methods for measurement of mRNA poly(A) tail length and heterogeneity. Poly(A) tail was first cleaved from mRNA with the RNase T1 enzyme. The average length of a liberated poly(A) tail was analyzed with the size exclusion chromatography method. Size heterogeneity of the poly(A) tail was estimated with high-resolution ion-pair reversed phase liquid chromatography (IP RP LC). The IP RP LC method provides resolution of poly(A) tail oligonucleotide variants up to 150 nucleotide long. Both methods use a robust ultraviolet detection suitable for mRNA analysis in quality control laboratories. The results were confirmed by the LC-mass spectrometry (LC MS) analysis of the same mRNA sample. The poly(A) tail length and heterogeneity results were in good agreement

Measurement and Modeling of Extra-Column Effects Due to Injection and Connections in Capillary Liquid Chromatography

As column volumes continue to decrease, extra-column band broadening has become an increasingly important consideration when determining column performance. Combined contributions due to the injector and connecting tubing in a capillary LC system were measured and found to be larger than expected by Taylor-Aris theory. Variance from sigma-type and tau-type broadening was isolated from eluted peaks using the Foley-Dorsey Exponentially Modified Gaussian peak fitting model and confirmed with computational fluid dynamics. It was found that the tau-type contributions were the main cause for the excessive broadening because of poorly-swept volumes at the connection between the injector and tubing. To reduce tau-type contributions (and peak tailing), a timed pinch mode could be used for analyte injection

Gebler, Implications of column peak capacity on the separation of complex peptide mixtures in single- and two-dimensional high-performance liquid chromatography

Abstract Column peak capacity was utilized as a measure of column efficiency for gradient elution conditions. Peak capacity was evaluated experimentally for reversed-phase (RP) and cation-exchange high-performance liquid chromatography (HPLC) columns, and compared to the values predicted from RP-HPLC gradient theory. The model was found to be useful for the prediction of peak capacity and productivity in single-and two-dimensional (2D) chromatography. Both theoretical prediction and experimental data suggest that the number of peaks separated in HPLC reaches an upper limit, despite using highly efficient columns or very shallow gradients. The practical peak capacity value is about several hundred for state-of-the-art RP-HPLC columns. Doubling the column length (efficiency) improves the peak capacity by only 40%, and proportionally increases both the separation time and the backpressure. Similarly, extremely shallow gradients have a positive effect on the peak capacity, but analysis becomes unacceptably long. The model predicts that a 2D-HPLC peak capacity of 15,000 can be achieved in 8 h using multiple fraction collection in the first dimension followed by fast RP-HPLC gradients employing short, but efficient columns in the second dimension

Insight into Trypsin Miscleavage: Comparison of Kinetic Constants of Problematic Peptide Sequences

Trypsin, a high fidelity protease, is the most widely used enzyme for protein digestion in proteomic research. Optimal digestion conditions are well-known and so are the expected cleavage products. However, missed cleavage sites are frequently observed when acidic amino acids, aspartic and glutamic acids, are present near the cleavage site. Also, the sequence motifs with successive lysine and/or arginine residues represent a source of missed cleaved sites. In spite of an adverse role of missed cleaved peptides on proteomic research, the digestion kinetics of these problematic sequences is not well-known. In this work, synthetic peptides with various sequence motifs were used as trypsin substrates. Cleavage products were analyzed with reversed-phase high performance liquid chromatography, and the kinetic constants for selected missed cleavage sites were calculated. Relative digestion speed for lysine and arginine sites is compared, including the digestion motifs flanked with aspartic and glutamic acid. Our findings show that DK and DTR motifs are cleaved by trypsin with 3 orders of magnitude lower speed than the arginine site. These motifs are likely to produce missed cleavage peptides in protein tryptic digests even at prolonged digestion times