V. V. Evreinov

Applicability of the Critical Chromatography Concept to Proteomics Problems : Experimental Study of the Dependence of Peptide Retention Time on the Sequence of Amino Acids in the Chain

Experimental data on the separation of synthetic and natural peptides are presented as treated in terms of the separation model proposed by the authors, which allows for the chain connectivity of amino acid residues and the cooperative character of their interaction with the surface. It was shown that the model accurately predicts the separation of peptides with identical amino acid contents and different sequences of units in the chain. The differences in the sequence may be permutation of amino acid residues and the presence of terminal groups, amino acid isomers, or mirror sequences in the chain. The separation model was used to predict the retention times of peptides prepared via the enzymatic hydrolysis of E. coli proteins and bovine serum albumin with trypsin. It was shown that in general the model accurately explains the array of experimental data on the separation of such peptides, thus being the first successful attempt to relate the chain sequence to the retention volume.

Inversion of chromatographic elution orders of peptides and its importance for proteomics

Inversion of the order of peptide elution in reversed-phase liquid chromatography under changing separation conditions, such as gradient slope has been considered. Using a six-protein proteolytic peptide standard and available literature data, the occurrence frequency and importance of this phenomenon in proteomic studies utilizing methods of shotgun proteomics and accurate mass and time tags have been evaluated. Feasibility of qualitative and quantitative description of peptide elution order inversion has been demonstrated using a model of critical liquid chromatography. Existing approaches to predict peptide separation and directions of the shifts of chromatographic peaks when the gradient profile changes have been compared.

Application of Statistical Thermodynamics To Predict the Adsorption Properties of Polypeptides in Reversed-Phase HPLC

The theory of critical chromatography for biomacromolecules (BioLCCC) describes polypeptide retention in reversed-phase HPLC using the basic principles of statistical thermodynamics. However, whether this theory correctly depicts a variety of empirical observations and laws introduced for peptide chromatography over the last decades remains to be determined. In this study, by comparing theoretical results with experimental data, we demonstrate that the BioLCCC: (1) fits the empirical dependence of the polypeptide retention on the amino acid sequence length with R(2) > 0.99 and allows in silico determination of the linear regression coefficients of the log-length correction in the additive model for arbitrary sequences and lengths and (2) predicts the distribution coefficients of polypeptides with an accuracy from 0.98 to 0.99 R(2). The latter enables direct calculation of the retention factors for given solvent compositions and modeling of the migration dynamics of polypeptides separated under isocratic or gradient conditions. The obtained results demonstrate that the suggested theory correctly relates the main aspects of polypeptide separation in reversed-phase HPLC.

Critical chromatography of macromolecules as a tool for reading the amino acid sequence of biomacromolecules: Reality or science fiction?

The capabilities of critical chromatography to study the amino acid sequences in biopolymer, peptide, and protein macromolecules are discussed. The rearrangement of two or more amino acid residues and the occurrence and location of modified residues in a chain are considered. The mechanism of an inversion in the order of peptide elution under changes in the gradient of the solvent composition is discussed.

Applicability of the critical-chromatography concept to analysis of proteins: Dependence of retention times on the sequence of amino acid residues in a chain

The BioLCCC model of the chromatographic separation of biomacromolecules, which involves the concepts of the critical chromatography of polymers, is used to describe the experimental data on the separation of proteins on different chromatographic systems. Using phenomenological parameters, i.e., effective adsorption energies of amino acid residues, we predict the effect of the sequence of these residues in the chain on retention times of proteins for reversed phases of different types (C4, C8, C18) in the gradient of a water-acetonitrile binary solvent. It is shown that, in general, the BioLCCC model correctly represents experimental data on the separation of proteins and makes it possible to quantitatively determine the effect of the sequence of amino acid residues on the separation. We show the limits of applicability of the model and explain the universal linear dependence that relates the retention volume and the logarithm of chain length that is observed in the chromatography of peptides and proteins.

Applicability of the critical chromatography concept to proteomics problems: I. Effect of the stationary phase and the size of the chromatographic column on the dependence of the retention time of peptides and proteins on the amino acid sequence

A theoretical study of the effect of stationary phase parameters on the regularities of the separation of peptides and proteins using a model of critical chromatography BioLCCC and the Theoretical chromatography software created on its basis has been performed. The following problems have been discussed: mechanism of the migration of chromatographic peak along the column and its compression in gradient elution; dependence of the coefficient of protein separation on the column length; inversion of the elution order of peptides at a change of the diameter or length of the column, adsorbent nature, conditions of emergence of inversion; and anomaly of the dependence of the retention time of proteins on the adsorbent activity.

On the utility of predictive chromatography to complement mass spectrometry based intact protein identification

AbstractThe amino acid sequence determines the individual protein three-dimensional structure and its functioning in an organism. Therefore, “reading” a protein sequence and determining its changes due to mutations or post-translational modifications is one of the objectives of proteomic experiments. The commonly utilized approach is gradient high-performance liquid chromatography (HPLC) in combination with tandem mass spectrometry. While serving as a way to simplify the protein mixture, the liquid chromatography may be an additional analytical tool providing complementary information about the protein structure. Previous attempts to develop “predictive” HPLC for large biomacromolecules were limited by empirically derived equations based purely on the adsorption mechanisms of the retention and applicable to relatively small polypeptide molecules. A mechanism of the large biomacromolecule retention in reversed-phase gradient HPLC was described recently in thermodynamics terms by the analytical model of liquid chromatography at critical conditions (BioLCCC). In this work, we applied the BioLCCC model to predict retention of the intact proteins as well as their large proteolytic peptides separated under different HPLC conditions. The specific aim of these proof-of-principle studies was to demonstrate the feasibility of using “predictive” HPLC as a complementary tool to support the analysis of identified intact proteins in top-down, middle-down, and/or targeted selected reaction monitoring (SRM)-based proteomic experiments. FigureIntact protein LC retention time prediction assists protein identification in top- and middle-down proteomics

Functionality-type distribution of poly(ethylene oxide) and poly(propylene oxide) macromonomers: Critical-chromatography study

Functionality-type distributions of macromomoners with poly(ethylene oxide) and poly(propylene oxide) chains are studied by chromatography under critical conditions. It is shown that, in the critical separation mode, separation of macromolecules with respect to size disappears and only information on the functionality-type distributions of the test samples may be derived. The critical conditions are determined experimentally with a normal phase (unmodified silica gel) for poly(propylene oxide) and with a reversed phase C18 for poly(ethylene oxide). The experimental retention volumes for bifunctional macromolecules are in satisfactory agreement with the values calculated under approximation of the Gaussian chain model.

Applicability of the critical-chromatography concept to proteomics problems: Separation of peptides modeled by a heterogeneous rod

The problems of separation of short peptides are considered in terms of the model of rigid rod adsorption. Analytical expressions relating the retention volume to the sequence of amino-acid residues in the gradient elution are obtained. The model of adsorption of a peptide as a rigid rod is compared with the model of its adsorption as a random-walk chain. The transition of rodlike peptides to the adsorbed state is more abrupt compared with the random-walk chain having the same sequence, while with an increase in the peptide length the random-walk chain model becomes more accurate. It is shown that the model of adsorption of a peptide as a rigid rod for short peptides fits the experimental data and correctly predicts the character of separation of peptides having equal lengths and identical amino-acid compositions but slightly differing in the alternation of residues in a chain.

Analysis of functionality-type distributions as a method for studying the kinetics of chemical reactions involving reactive oligomers

Critical chromatography is used to study the kinetics of reactions between polypropylene glycols and phenyl butyl isocyanate that simulate the production of polyester urethanes. The use of a two-detector version of chromatography makes it possible to monitor the concentration of the original, intermediate, and final oligomers and reveal the effect of one end group on the reactivity of the other separated by a long chain. The processing of the kinetic data makes it possible to determine the reaction-rate constants for macromolecules of different functionalities and to quantitatively describe the process behavior.