Marina L. Pridatchenko

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.

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.

Depletion of human serum albumin in embryo culture media for in vitro fertilization using monolithic columns with immobilized antibodies.

Affinity depletion of abundant proteins such as HSA is an important stage in routine sample preparation prior to MS/MS analysis of biological samples with high range of concentrations. Due to the charge competition effects in electrospray ion source that results in discrimination of the low‐abundance species, as well as limited dynamic range of MS/MS, restricted typically by three orders of magnitude, the identification of low‐abundance proteins becomes a challenge unless the sample is depleted from high‐concentration compounds. This dictates a need for developing efficient separation technologies allowing fast and automated protein depletion. In this study, we performed evaluation of a novel immunoaffinity‐based Convective Interaction Media analytical columns (CIMac) depletion column with specificity to HSA (CIMac‐αHSA). Because of the convective flow‐through channels, the polymethacrylate CIMac monoliths afford flow rate independent binding capacity and resolution that results in relatively short analysis time compared with traditional chromatographic supports. Seppro IgY14 depletion kit was used as a benchmark to control the results of depletion. Bottom‐up proteomic approach followed by label‐free quantitation using normalized spectral indexes were employed for protein quantification in G1/G2 and cleavage/blastocyst in vitro fertilization culture media widely utilized in clinics for embryo growth in vitro. The results revealed approximately equal HSA level of 100 ± 25% in albumin‐enriched fractions relative to the nondepleted samples for both CIMac‐αHSA column and Seppro kit. In the albumin‐free fractions concentrated 5.5‐fold by volume, serum albumin was identified at the levels of 5–30% and 20–30% for the CIMac‐αHSA and Seppro IgY14 spin columns, respectively.

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.

Ion coalescence in Fourier transform mass spectrometry: should we worry about this in shotgun proteomics?

Coupling of motion of the ion clouds with close m/z values is a well-established phenomenon for ion-trapping mass analyzers. In Fourier transform ion cyclotron resonance mass spectrometry it is known as ion coalescence. Recently, ion coalescence was demonstrated and semiquantitatively characterized for the Orbitrap mass analyzer as well. When it occurs, the coalescence negatively affects the basic characteristics of a mass analyzer. Specifically, the dynamic range available for the high resolving power mass measurements reduces. In shotgun proteomics, another potentially adverse effect of ion coalescence is interference of the isotopic envelopes for the coeluting precursor ions of close m/z values, subjected to isolation before fragmentation. In this work we characterize coalescence events for synthetic peptide mixtures with fully and partially overlapping 13C-isotope envelopes, including pairs of peptides with glutamine deamidation. Furthermore, we demonstrate that fragmentation of the otherwise coalesced peptide ion clouds may remove the locking between them owing to the total charge redistribution between more ion species in the mass spectrum. Finally, we estimated the possible scale of the coalescence phenomenon for shotgun proteomics by considering the fraction of coeluted peptide pairs with the close masses using literature data for the yeast proteome. It was found that up to one-tenth of all peptide identifications with the relative mass differences of 20 ppm or less in the corresponding pairs may potentially experience the coalescence of the 13C-isotopic envelopes. However, sample complexity in a real proteomics experiment and precursor ion-signal splitting between many channels in tandem mass spectrometry drastically increase the threshold for coalescence, thus leading to practically coalescence-free proteomics based on Fourier transform mass spectrometry.

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

Peptide identification in “shotgun” proteomics using tandem mass spectrometry: Comparison of search engine algorithms

High-throughput proteomics technologies are gaining popularity in different areas of life sciences. One of the main objectives of proteomics is characterization of the proteins in biological samples using liquid chromatography/mass spectrometry analysis of the corresponding proteolytic peptide mixtures. Both the complexity and the scale of experimental data obtained even from a single experimental run require specialized bioinformatic tools for automated data mining. One of the most important tools is a so-called proteomics search engine used for identification of proteins present in a sample by comparing experimental and theoretical tandem mass spectra. The latter are generated for the proteolytic peptides derived from a protein database. Peptide identifications obtained with the search engine are then scored according to the probability of a correct peptide-spectrum match. The purpose of this work was to perform a comparison of different search algorithms using data acquired for complex protein mixtures, including both annotated protein standards and clinical samples. The comparison was performed for three popular search engines: commercially available Mascot, as well as open-source X!Tandem and OMSSA. It was shown that the search engine OMSSA identifies in general a smaller number of proteins, while X!Tandem and Mascot deliver similar performance. We found no compelling reasons for using the commercial search engine instead of its open source competitor.

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.