I. A. Tarasova

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: Dependence of retention time on the sequence of amino acids

A peptide separation model based on the technique of liquid chromatography of macromolecules at the critical condition was proposed. In terms of this model, the array of experimental data on the separation of peptides is considered. The main phenomenological parameters of the model—effective adsorption energies of amino acid residues—were determined, thus allowing the influence of character of their alternation in the chain on retention times to be predicted. The model is applicable to investigation into the feasibility of separation in different chromatographic modes of not only peptides with the same amino acid composition and different sequences of units in the chain but also peptides containing amino acid isomers and mirror sequences with different terminal groups.

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.

Probing the mechanisms of ambient ionization by laser‐induced fluorescence spectroscopy

The ionization mechanisms of several atmospheric pressure ion sources based on desorption and ionization of samples deposited on a surface were studied. Home-built desorption electrospray ionization (DESI), laserspray ionization (LSI), and atmospheric pressure matrix-assisted laser desorption/ionization (AP-MALDI) sources were characterized using low-molecular-weight compounds, in particular fluorescent dyes. Detection of the desorbed and ionized species was performed by laser-induced fluorescence and ion cyclotron resonance mass spectrometry. The dependences of the signal intensities on various experimental parameters were studied. The data obtained reveals common features, such as formation of solvated species and clusters in the ionization processes, in all of the techniques considered.

Empirical approach to false discovery rate estimation in shotgun proteomics

Estimation of false discovery rate (FDR) for identified peptides is an important step in large-scale proteomic studies. We introduced an empirical approach to the problem that is based on the FDR-like functions of sets of peptide spectral matches (PSMs). These functions have close values for equal-sized sets with the same FDR and depend monotonically on the FDR of a set. We have found three of them, based on three complementary sources of data: chromatography, mass spectrometry, and sequences of identified peptides. Using a calibration on a set of putative correct PSMs these functions were converted into the FDR scale. The approach was tested on a set of approximately 2800 PSMs obtained from rat kidney tissue. The estimates based on all three data sources were rather consistent with each other as well as with one made using the target-decoy strategy.

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.

Comparative evaluation of label‐free quantification methods for shotgun proteomics

RATIONALE Label-free quantification (LFQ) is a popular strategy for shotgun proteomics. A variety of LFQ algorithms have been developed recently. However, a comprehensive comparison of the most commonly used LFQ methods is still rare, in part due to a lack of clear metrics for their evaluation and an annotated and quantitatively well-characterized data set. METHODS Five LFQ methods were compared: spectral counting based algorithms SIN , emPAI, and NSAF, and approaches relying on the extracted ion chromatogram (XIC) intensities, MaxLFQ and Quanti. We used three criteria for performance evaluation: coefficient of variation (CV) of protein abundances between replicates; analysis of variance (ANOVA); and the root-mean-square error of logarithmized calculated concentration ratios, referred to as standard quantification error (SQE). Comparison was performed using a quantitatively annotated publicly available data set. RESULTS The best results in terms of inter-replicate reproducibility were observed for MaxLFQ and NSAF, although they exhibited larger standard quantification errors. Using NSAF, all quantitatively annotated proteins were correctly identified in the Bonferronni-corrected results of the ANOVA test. SIN was found to be the most accurate in terms of SQE. Finally, the current implementations of XIC-based LFQ methods did not outperform the methods based on spectral counting for the data set used in this study. CONCLUSIONS Surprisingly, the performances of XIC-based approaches measured using three independent metrics were found to be comparable with more straightforward and simple MS/MS-based spectral counting approaches. The study revealed no clear leader among the latter. Copyright

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.

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.