DirectMS1: MS/MS-Free Identification of 1000 Proteins of Cellular Proteomes in 5 Minutes.

Proteome characterization relies heavily on tandem mass spectrometry (MS/MS) and is thus associated with instrumentation complexity, lengthy analysis time, and limited duty cycle. It was always tempting to implement approaches that do not require MS/MS, yet they were constantly failing to achieve a meaningful depth of quantitative proteome coverage within short experimental times, which is particularly important for clinical or biomarker-discovery applications. Here, we report on the first successful attempt to develop a truly MS/MS-free method, DirectMS1, for bottom-up proteomics. The method is compared with the standard MS/MS-based data-dependent acquisition approach for proteome-wide analysis using 5 min LC gradients. Specifically, we demonstrate identification of 1 000 protein groups for a standard HeLa cell line digest. The amount of loaded sample was varied in a range from 1 to 500 ng, and the method demonstrated 10-fold higher sensitivity. Combined with the recently introduced Diffacto approach for relative protein quantification, DirectMS1 outperforms most popular MS/MS-based label-free quantitation approaches because of significantly higher protein sequence coverage.

An Internally Quenched Fluorescent Peptide Substrate for Protealysin.

Protealysin, a metalloprotease of Serratia proteamaculans, is the prototype of a subgroup of the M4 peptidase family. Protealysin-like proteases (PLPs) are widely spread in bacteria but also occur in fungi and certain archaea. The interest in PLPs is primarily due to their putative involvement in the bacterial pathogenesis in animals and plants. Studying PLPs requires an efficient quantitative assay for their activity; however, no such assay has been reported so far. Here, we used the autoprocessing site sequence of the protealysin precursor to construct an internally quenched fluorescent peptide substrate 2-aminobenzoyl-L-arginyl-L-seryl-L-valyl-L-isoleucyl-L-(ε-2,4-dinitrophenyl)lysine. Protealysin and thermolysin, the prototype of the M4 family, proved to hydrolyze only the Ser-Val bond of the substrate. The substrate exhibited a KM = 35 ± 4 µM and kcat = 21 ± 1 s-1 for protealysin as well as a KM = 33 ± 8 µM and kcat = 7 ± 1 s-1 for thermolysin at 37 °C. Comparison of the effect of different enzymes (thermolysin, trypsin, chymotrypsin, savinase, and pronase E) on the substrate has demonstrated that it is not strictly specific for protealysin; however, this enzyme has higher molar activity even compared to the closely related thermolysin. Thus, the proposed substrate can be advantageous for quantitative studies of protealysin as well as for activity assays of other M4 peptidases.

IdentiPy: An Extensible Search Engine for Protein Identification in Shotgun Proteomics.

We present an open-source, extensible search engine for shotgun proteomics. Implemented in Python programming language, IdentiPy shows competitive processing speed and sensitivity compared with the state-of-the-art search engines. It is equipped with a user-friendly web interface, IdentiPy Server, enabling the use of a single server installation accessed from multiple workstations. Using a simplified version of X!Tandem scoring algorithm and its novel "autotune" feature, IdentiPy outperforms the popular alternatives on high-resolution data sets. Autotune adjusts the search parameters for the particular data set, resulting in improved search efficiency and simplifying the user experience. IdentiPy with the autotune feature shows higher sensitivity compared with the evaluated search engines. IdentiPy Server has built-in postprocessing and protein inference procedures and provides graphic visualization of the statistical properties of the data set and the search results. It is open-source and can be freely extended to use third-party scoring functions or processing algorithms and allows customization of the search workflow for specialized applications.

MS/MS-Free Protein Identification in Complex Mixtures Using Multiple Enzymes with Complementary Specificity.

In this work, we present the results of evaluation of a workflow that employs a multienzyme digestion strategy for MS1-based protein identification in "shotgun" proteomic applications. In the proposed strategy, several cleavage reagents of different specificity were used for parallel digestion of the protein sample followed by MS1 and retention time (RT) based search. Proof of principle for the proposed strategy was performed using experimental data obtained for the annotated 48-protein standard. By using the developed approach, up to 90% of proteins from the standard were unambiguously identified. The approach was further applied to HeLa proteome data. For the sample of this complexity, the proposed MS1-only strategy determined correctly up to 34% of all proteins identified using standard MS/MS-based database search. It was also found that the results of MS1-only search were independent of the chromatographic gradient time in a wide range of gradients from 15-120 min. Potentially, rapid MS1-only proteome characterization can be an alternative or complementary to the MS/MS-based "shotgun" analyses in the studies, in which the experimental time is more important than the depth of the proteome coverage.

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.

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 (13)C-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 (13)C-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.

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.

Empirical multidimensional space for scoring peptide spectrum matches in shotgun proteomics.

Data-dependent tandem mass spectrometry (MS/MS) is one of the main techniques for protein identification in shotgun proteomics. In a typical LC-MS/MS workflow, peptide product ion mass spectra (MS/MS spectra) are compared with those derived theoretically from a protein sequence database. Scoring of these matches results in peptide identifications. A set of peptide identifications is characterized by false discovery rate (FDR), which determines the fraction of false identifications in the set. The total number of peptides targeted for fragmentation is in the range of 10,000 to 20,000 for a several-hour LC-MS/MS run. Typically, <50% of these MS/MS spectra result in peptide-spectrum matches (PSMs). A small fraction of PSMs pass the preset FDR level (commonly 1%) giving a list of identified proteins, yet a large number of correct PSMs corresponding to the peptides originally present in the sample are left behind in the "grey area" below the identity threshold. Following the numerous efforts to recover these correct PSMs, here we investigate the utility of a scoring scheme based on the multiple PSM descriptors available from the experimental data. These descriptors include retention time, deviation between experimental and theoretical mass, number of missed cleavages upon in-solution protein digestion, precursor ion fraction (PIF), PSM count per sequence, potential modifications, median fragment mass error, (13)C isotope mass difference, charge states, and number of PSMs per protein. The proposed scheme utilizes a set of metrics obtained for the corresponding distributions of each of the descriptors. We found that the proposed PSM scoring algorithm differentiates equally or more efficiently between correct and incorrect identifications compared with existing postsearch validation approaches.

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

The 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.

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.