A. V. Gorshkov

Retention time prediction using the model of liquid chromatography of biomacromolecules at critical conditions in LC-MS phosphopeptide analysis

LC combined with MS/MS analysis of complex mixtures of protein digests is a reliable and sensitive method for characterization of protein phosphorylation. Peptide retention times (RTs) measured during an LC‐MS/MS run depend on both the peptide sequence and the location of modified amino acids. These RTs can be predicted using the LC of biomacromolecules at critical conditions model (BioLCCC). Comparing the observed RTs to those obtained from the BioLCCC model can provide additional validation of MS/MS‐based peptide identifications to reduce the false discovery rate and to improve the reliability of phosphoproteome profiling. In this study, energies of interaction between phosphorylated residues and the surface of RP separation media for both “classic” alkyl C18 and polar‐embedded C18 stationary phases were experimentally determined and included in the BioLCCC model extended for phosphopeptide analysis. The RTs for phosphorylated peptides and their nonphosphorylated analogs were predicted using the extended BioLCCC model and compared with their experimental RTs. The extended model was evaluated using literary data and a complex phosphoproteome data set distributed through the Association of Biomolecular Resource Facilities Proteome Informatics Research Group 2010 study. The reported results demonstrate the capability of the extended BioLCCC model to predict RTs which may lead to improved sensitivity and reliability of LC‐MS/MS‐based phosphoproteome profiling.

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.

Limitation of predictive 2‐D liquid chromatography in reducing the database search space in shotgun proteomics: In silico studies

A two-dimensional (2-D) liquid chromatography (LC) separation of complex peptide mixtures that combines a normal phase utilizing hydrophilic interactions and a reversed phase offers reportedly the highest level of 2-D LC orthogonality by providing an even spread of peptides across multiple LC fractions. Matching experimental peptide retention times to those predicted by empirical models describing chromatographic separation in each LC dimension leads to a significant reduction in a database search space. In this work, we calculated the retention times of tryptic peptides separated in the C18 reversed phase at different separation conditions (pH 2 and pH 10) and in TSK gel Amide-80 normal phase. We show that retention times calculated for different 2-D LC separation schemes utilizing these phases start to correlate once the mass range of peptides under analysis becomes progressively narrow. This effect is explained by high degree of correlation between retention coefficients in the considered phases.

Do Oil-Degrading Rhodococci Contribute to the Genesis of Deep-Water Bitumen Mounds in Lake Baikal?

This study sought to understand the origin and fate of one of the bitumen mounds found on the bottom of Lake Baikal. These mounds are located at a depth of 900 m beneath oil spots detected on the surface of Lake Baikal (53° 18′24″, 108° 23′20″). The two mounds were sampled with a manipulator from a “MIR” deep-water manned submersible. Mature mound No. 8 was subjected to chemical and microbiological studies. Mound No. 3 was subjected only to chemical studies; we failed to perform microbiological analyses of this mound for logistic reasons. Oil spots collected from the water surface, samples of mound No. 3 and No. 8, were subjected to GC/MS analysis. The water contained aliphatic hydrocarbons with chains between C8 and C23, with the most abundant chain length being C18. Mound No. 3 with the most abundant chain length being C18 actively released oil droplets into the water. It contained 770 mg/g of C13-C32 n-alkanes, with a maximum at C23 (160 mg/g). Mound No. 8 was inactive and contained 148 mg/g of aliphatic C22-C34 n-alkanes, with a maximum at C25. Mound No. 8 also consisted of 3% inorganic matter, 48% unresolved complex mixture (UCM) and less than 1% other compounds (polyaromatic hydrocarbons, isoprenoids, carotenoids, and hopanes). The core of this sample used as inoculate, yielded Rhodococci when cultivated on oil as the only source of carbon. Cultivation of the sample on agar-containing Raymond inorganic medium with crude West Siberian oil as the only source of carbon revealed colonies of these bacteria, which all appeared identical. PCR was performed with DNA isolated from 5 colonies, using primers for 16S rRNA genes. Comparison of the sequences of the 5 PCR products over a length of 714 bp revealed that they were almost identical. Phylogenetic analysis of these homologous sequences showed that they were similar to the corresponding sequences of the genus Rhodococcus. Substrate demands, the morphology of the colonies, and SEM and TEM data confirmed that the isolates obtained could indeed be Rhodococci. All of the isolates could grow in bulk cultures with inorganic medium supplemented with crude oil. Moreover, all of the isolates degraded aliphatic hydrocarbons with lengths between C11 and C29. C23-C29 hydrocarbons were degraded completely. The isolates could grow at 4–37°C. The most unexpected finding was that of the many microorganisms capable of consuming oil, only Rhodococci exhibited this ability in the inactive bitumen mound. The possible mechanisms of how crude oil is transformed into bitumen mounds and mature bitumen are 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.

Green chemistry of polyurethanes: Synthesis, structure, and functionality of triglycerides of soybean oil with epoxy and cyclocarbonate groups—renewable raw materials for new urethanes

The synthesis of cyclocarbonate-containing oligomers for non-isocyanate polyurethanes via the catalytic reaction of carbon dioxide with epoxidized soybean oil has been described. It has been shown via the mass-spectrometric method that the oligomers, which are triglycerides of epoxide- and cyclocarbonate-containing derivatives of carboxylic acids, are characterized by an extremely wide composition distribution, reflecting almost the entire set of components contained in natural raw materials. The presence of 25 types of triglycerides of unsaturated (oleic, linoleic, and linolenic) and saturated (stearic and palmitic) carboxylic acids in various combinations in oligomers has been found. The functionality of oligomers at epoxy and cyclocarbonate groups and the problems of the use of plant raw materials for the synthesis of non-isocyanate polyurethanes are discussed.