The use of CRISPR/Cas nucleases for the development of DNA diagnostic systems in out-of-laboratory conditions (point-of-need testing, PONT) has demonstrated rapid growth in the last few years, starting with the appearance in 2017–2018 of the first diagnostic platforms known as DETECTR and SHERLOCK. The platforms are based on a combination of methods of nucleic acid isothermal amplification with selective CRISPR/Cas detection of target amplicons. This significantly improves the sensitivity and specificity of PONT, making them comparable with or even superior to the sensitivity and specificity of polymerase chain reaction, considered as the “gold standard” of DNA diagnostics. The review considers modern approaches to the coupling of CRISPR/Cas detection using Cas9, Cas12a, Cas12b, Cas13a, Cas14, and Cas3 nucleases to various methods of nucleic acid isothermal amplification, with an emphasis on works in which sensitivity at the level of single molecules (attomolar and subattomolar concentrations of the target) is achieved. The properties of CRISPR/Cas nucleases used for targeted DNA diagnostics and the features of methods of nucleic acid isothermal amplification are briefly considered in the context of the development of diagnostic biosensing platforms. Special attention is paid to the most promising directions for the development of DNA diagnostics using CRISPR/Cas nuclease.
A workshop was convened in Moscow by the Human Proteome Organization on March 2009 to review the current status of the Human Proteome Project (HPP) 1The abbreviations used are:HPPHuman Proteome ProjectPTMpost-translational modificationMRMmultiple reaction monitoringAQUAAbsolute QuantitationPSAQprotein standard absolute quantification. 1The abbreviations used are:HPPHuman Proteome ProjectPTMpost-translational modificationMRMmultiple reaction monitoringAQUAAbsolute QuantitationPSAQprotein standard absolute quantification. and to discuss how this project could be most effectively launched. Presentations were delivered by representatives from 8 countries, including Russia, Canada, China, France, Japan, Korea, Sweden, the USA as well as the European Commission. Human Proteome Project post-translational modification multiple reaction monitoring Absolute Quantitation protein standard absolute quantification. Human Proteome Project post-translational modification multiple reaction monitoring Absolute Quantitation protein standard absolute quantification. A Russian focus on chromosome 18 was covered by Prof. Alexander Archakov with a proposal to use atomic force microscopy and the iterative irreversible association of biomolecules onto chemically active surfaces to characterize proteins at concentrations below 10−12 m. Support for the Russian contribution to the Human Proteome Project was extended by the Deputy Minister of Education and Science of the Russian Federation, Alexander Khlunov, and Anatoly Grigoriev, Vice-President of the Russian Academy of Sciences. Dr. Grigoriev proposed to extend a disease-centered paradigm to the variability of the proteome in the normal healthy person with support indicated by Alexander Gintsburg, Vice-President of the Russian Academy of Medical Sciences and a Member of the Presidential Council for Science and High Technologies, Academician Konstantin Skryabin. He indicated that Russia has already created technical and human resources for large-scale international projects. Workshop presentations and discussions revolved around a white paper of the HPP, put together during a Barbados workshop in January 2008 and finalized in August 2008. The vision of the white paper comprised three research engines, interfaced to each other at the bioinformatics and computational biology layer. The first engine defines the complete proteome through a mapping with antibodies. In this regard, Prof. F. Ponten (Uppsala University) revealed progress on the Human Protein Atlas Project. He also illustrated the clinical relevance of a systematic antibody mapping of the proteome. The core of the second engine depends upon mass spectrometry, and here many speakers shared the opinion of Dr. S. Hanash (Fred Hutchinson Cancer Research Center, USA) that the technology is already accurate enough to deliver for the medicinal sector. For example, within the International Disease Biomarker Project the cancer biomarkers requirements include risk assessment, early detection, molecular classification, and disease monitoring. However, Prof. R. Bradshaw (University of California, San Francisco) drew attention to the lack of biomarker identification to date and the fact that few if any validated biomarkers have resulted from proteomics experiments (1Zolg W. The proteomic search for diagnostic biomarkers: lost in translation?.Mol. Cell. Proteomics. 2006; 5: 1720-1726Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). He noted that technology may not yet be able to detect biomarkers in complex samples such as plasma, or worse, there may be no useful biomarkers to detect. On the positive side, Dr. N. Taniguchi from Osaka University illustrated the diagnostic power of core fucosylation of N-glycans using mass spectrometry and the fucose-binding Aleuria aurantia lectin. Even analyzing the proteins within the normal range of mass spectrometry detection (haptoglobin, alpha-fetoprotein, etc.), it was possible to unravel the differences associated with emphysema, pancreatic cancer, hepatoma, and liver cirrhosis by focusing on fucose modifications. The progress and already realized clinical applications of glycomics were compared with that of phosphoproteomics. As indicated by Dr. Ralph Bradshaw, even though protein phosphorylations represent a minor component of detectable post-translational modifications (PTMs), the literature is strongly biased toward the description of protein phosphorylation. The general scaffold of how a country can organize its portion of HPP duties in compliance to the joint efforts around the world was presented by the HUPO President Prof. Young Ki-Paik (Yonsei University, Korea). With chromosome 13 as an example, he navigated participants of the workshop through complementary research strategies, which spanned through a starting two-year period of the project with a workflow scheduled for 2010–2018. Again, three aspects of the gene-centric HPP constituted the core of the Korean approach: MS-based, antibody-based, and network-based engines. Strategy one covered multiple reaction monitoring (MRM)-based protein quantification, whereas strategy two also included hybrid antibodies at the protein enrichment stage. The problem of quantification was also an issue of the other reports, starting from the semi-quantitative approach practiced by the Human Liver Proteome Project (HLPP) chosen by Dr. P. Yang (Fudan University) and Absolute Quantitation (AQUA) chosen by KHUPO, and ending with a Protein Standard Absolute Quantification (PSAQ) method, which utilizes full-length isotope-labeled proteins. The latter approach was presented by Prof. Pierre Legrain (Commissariat à l'Energie Atomique, France) to demonstrate its preferences over the conventional protein quantification technologies such as AQUA and Quantification concatamers (QconCAT) for absolute quantification employing concatenated signature peptides of artificial proteins. The difficulty of protein quantification led to an appreciation as to how a gene-centric approach can be used to share the burden of HPP expenses among countries. Whether it is AQUA or PSAQ or any other, a single batch of labeled peptides or proteins may need to be explored by many labs. The price per protein coding gene becomes feasible in the case that the standards are available to many users. Approximate figures can be drawn from the paper by Anderson et al. (2Anderson N.L. Anderson N.G. Pearson T.W. Borchers C.H. Paulovich A.G. Patterson S.D. Gillette M. Aebersold R. Carr S.A. Human proteome detection and quantitation project.Mol. Cell. Proteomics. 2009; 5: 883-886Abstract Full Text Full Text PDF Scopus (172) Google Scholar), who launched the human Proteome Detection and Quantitation (hPDQ) initiative to enable individual researchers to measure defined collections of human proteins in biosamples. The price of isotopic assays of proteotypic peptides was assessed as 50 million USD for 2000 proteins and 250 million USD for all protein-coding genes. Such costs would be prohibitive for any one nation, but since any one nation could be responsible for only one chromosome, the expenses can be shared. This would imply the need for 24 nations with 22 for the autosomes and 2 for the sex chromosomes. In the course of round-table discussions, advances in peptidomic research were overviewed by Prof. V. Ivanov (Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences). The peptidome offers considerable promise for diagnostic purposes, as pathological processes may be linked to reproducible changes in the peptide composition of biofluids. Furthermore, the peptidome may become a source of biologically active peptides, which can be turned into pharmacological compounds (3Ivanov V.T. Yatskin O.N. Peptidomics: a logical sequel to proteomics.Expert. Rev. Proteomics. 2005; 2: 463-473Crossref PubMed Scopus (51) Google Scholar). Projects in human health and disease will likely articulate a protein-centric view of genes as a complement to the gene-centric view of proteins (4Hochstrasser D. Should the human proteome project be gene- or protein-centric?.J. Proteome Res. 2008; 7: 5071Crossref PubMed Scopus (8) Google Scholar). That would be compliant with the disease-related mission of the project. The importance of quantitative analysis in the development of proteomics was emphasized by Prof. R. Bradshaw who indicated, “In proteomics, we generally collect snapshots, whereas in reality the proteome is a movie. To bridge this gap, quantitative measurements are and will be essential and this is the future direction of much of proteomics research. Within two years it will likely become the norm rather than the exception”. In this regard, the recent Clinical Proteomic Technology Assessment for Cancer (CPTAC) study (5Addona T.A. Abbatiello S.E. Schilling B. Skates S.J. Mani D.R. Bunk D.M. Spiegelman C.H. Zimmerman L.J. Ham A.J. Keshishian H. Hall S.C. Allen S. Blackman R.K. Borchers C.H. Buck C. Cardasis H.L. Cusack M.P. Dodder N.G. Gibson B.W. Held J.M. Hiltke T. Jackson A. Johansen E.B. Kinsinger C.R. Li J. Mesri M. Neubert T.A. Niles R.K. Pulsipher T.C. Ransohoff D. Rodriguez H. Rudnick P.A. Smith D. Tabb D.L. Tegeler T.J. Variyath A.M. Vega-Montoto L.J. Wahlander A. Waldemarson S. Wang M. Whiteaker J.R. Zhao L. Anderson N.L. Fisher S.J. Liebler D.C. Paulovich A.G. Regnier F.E. Tempst P. Carr S.A. Multi-site assessment of the precision and reproducibility of multiple reaction monitoring-based measurements of proteins in plasma.Nat. Biotechnol. 2009; 27: 633-641Crossref PubMed Scopus (819) Google Scholar) on the reproducibility of multiple reaction monitoring based measurements of proteins in plasma complements the HUPO test sample study (6Bell A.W. Deutsch E.W. Au C.E. Kearney R.E. Beavis R. Sechi S. Nilsson T. Bergeron J.J.M. HUPO Test Sample Working Group A HUPO test sample study reveals common problems in mass spectrometry-based proteomics.Nat. Methods. 2009; 6: 423-430Crossref PubMed Scopus (259) Google Scholar) to demonstrate reproducibility and accuracy of characterization of test sample proteins. The thesis of “no plasma - no money” (Dr. S. Hanash) was indicated, but whether all of the proteins actually reside in the plasma is unknown. To populate genes with MS spectra, it is necessary to find the preferential residence of desired proteins by antibody mapping of protein localizations as deduced from the predicted protein coding human genes. After that, inspection of a particular cellular subfraction may have a greater chance to access proteins rather than plasma. No dedicated database for the HPP exists today. It was proposed that HPP database construction will be needed under the supervision of HUPO. This database would follow up the gene-centric principle as a convenient scaffold to assemble the data produced by the HPP research engines. The idea behind such a database is to create the global source, which anyone can access for an update, for knowledge that emanates from the HPP. This database will serve as a central warehouse to collect data from the gene-centric databases for antibody and mass spectrometry-based proteomics. We thank Dr. Gil Omenn (University of Michigan), Chair of HUPO Initiatives, for his helpful comments.
Proteome profiling of human testicular biopsies was performed using tandem mass spectrometry with electrospray ionization. Protein identification results were compared for the Mascot commercial search engine, the SearchGUI noncommercial package, and their analog IdentiProt based on the open-source IdentiPy algorithm (http://hg.theorchromo.ru/identipy). A feature of IdentiPy is an automatic optimization of MS/MS search parameters. A set of protein identifications obtained with IdentiPy was consequently greater by one third than the sets with the other search engines. For the first time, an IdentiPy/IdentiProt search was conducted within the Progenesis LC-MS framework, which allows spectrum alignment, and the proteome profile obtained with alignment was compared with that obtained using the ProteoWizard converter. A total of 16 human chromosome 18 proteins were identified, including the myelin basic protein, which is not characteristic of testicular tissue.
The article summarizes the achievements of the pilot phase (2010-2014) of the Russian part of the international project “Human Proteome” and identifies the directions for further work on the study of the human chromosome 18 proteome in the framework of the project main phase (2015-2022). The pilot phase of the project was focused on the detection of at least one protein for each chromosome 18 protein-coding gene in three types of the biological material. The application of mass spectrometric detection of proteins by the methods of multiple reactions monitoring (MRM) and gene-centric approach made it possible to detect 95% of master forms of proteins, for 60% of which the quantitative assessment of the protein content was obtained in at least one type of the biological material. The task of the main phase of the project is to measure the proteome size of healthy individuals, taking into account the modified protein forms, providing for both the bioinformatics prediction of the quantity of proteins types and the selective experimental measurement of single proteoforms. Since the ranges of protein concentrations corresponding to the normal physiological state have not been identified, the work of the main phase of the project is focused on the study of clinically healthy individuals. The absence of these data complicates significantly the interpretation of the patients’ blood proteomic profiles and prevents creating diagnostic tests. In the long term prospect, implementation of the project envisages development of a diagnostic test system based on multiple reactions monitoring (MRM) for quantitative measurement of the protein forms associated with some diseases. Development of such test systems will allow predicting the extent of risk of different diseases, diagnosing a disease at its early stage and monitoring the effectiveness of the treatment.
This work was aimed at estimating the concentrations of proteins encoded by human chromosome 18 (Chr 18) in plasma samples of 54 healthy male volunteers (aged 20-47). These young persons have been certified by the medical evaluation board as healthy subjects ready for space flight training. Over 260 stable isotope-labeled peptide standards (SIS) were synthesized to perform the measurements of proteins encoded by Chr 18. Selected reaction monitoring (SRM) with SIS allowed an estimate of the levels of 84 of 276 proteins encoded by Chr 18. These proteins were quantified in whole and depleted plasma samples. Concentration of the proteins detected varied from 10-6 M (transthyretin, P02766) to 10-11 M (P4-ATPase, O43861). A minor part of the proteins (mostly representing intracellular proteins) was characterized by extremely high inter individual variations. The results provide a background for studies of a potential biomarker in plasma among proteins encoded by Chr 18. The SRM raw data are available in ProteomeXchange repository (PXD004374).
Proteomic challenges, stirred up by the advent of high-throughput technologies, produce large amount of MS data. Nowadays, the routine manual search does not satisfy the "speed" of modern science any longer. In our work, the necessity of single-thread analysis of bulky data emerged during interpretation of HepG2 proteome profiling results for proteoforms searching. We compared the contribution of each of the eight search engines (X!Tandem, MS-GF[Formula: see text], MS Amanda, MyriMatch, Comet, Tide, Andromeda, and OMSSA) integrated in an open-source graphical user interface SearchGUI ( http://searchgui.googlecode.com ) into total result of proteoforms identification and optimized set of engines working simultaneously. We also compared the results of our search combination with Mascot results using protein kit UPS2, containing 48 human proteins. We selected combination of X!Tandem, MS-GF[Formula: see text] and OMMSA as the most time-efficient and productive combination of search. We added homemade java-script to automatize pipeline from file picking to report generation. These settings resulted in rise of the efficiency of our customized pipeline unobtainable by manual scouting: the analysis of 192 files searched against human proteome (42153 entries) downloaded from UniProt took 11[Formula: see text]h.
Determination of protein concentration in biological samples is an important task for biological research, as well as for medicine and routine clinical biochemistry. The introduction of stable isotope-labeled peptide standards (SIS) made it possible to determine accurately absolute protein concentrations in proteomic studies. The correct choice of SIS and the systematic way to develop a s method for selected reaction monitoring (SRM) are very important steps that are crucial for further identification and measurements of protein concentration. In this paper, we summarize our experience of selecting SIS for measuring the protein concentration by SRM. The results are presented in the form of an algorithm that describes the main stages of the SIS selection and the main points in the development of SRM methods for the targeted protein detection and determination of protein concentrations in biological samples.
Background: Colorectal cancer (CRC) at a current clinical level is still hardly diagnosed, especially with regard to nascent tumors, which are typically asymptotic. Searching for reliable biomarkers of early diagnosis is an extremely essential task. Identification of specific post-translational modifications (PTM) may also significantly improve net benefits and tailor the process of CRC recognition. We examined depleted plasma samples obtained from 41 healthy volunteers and 28 patients with CRC at different stages to conduct comparative proteome-scaled analysis. The main goal of the study was to establish a constellation of protein markers in combination with their PTMs and semi-quantitative ratios that may support and realize the distinction of CRC until the disease has a poor clinical manifestation. Results: Proteomic analysis revealed 119 and 166 proteins for patients in stages I–II and III–IV, correspondingly. Plenty of proteins (44 proteins) reflected conditions of the immune response, lipid metabolism, and response to stress, but only a small portion of them were significant (p < 0.01) for distinguishing stages I–II of CRC. Among them, some cytokines (Clusterin (CLU), C4b-binding protein (C4BP), and CD59 glycoprotein (CD59), etc.) were the most prominent and the lectin pathway was specifically enhanced in patients with CRC. Significant alterations in Inter-alpha-trypsin inhibitor heavy chains (ITIH1, ITIH2, ITIH3, and ITIH4) levels were also observed due to their implication in tumor growth and the malignancy process. Other markers (Alpha-1-acid glycoprotein 2 (ORM2), Alpha-1B-glycoprotein (A1BG), Haptoglobin (HP), and Leucine-rich alpha-2-glycoprotein (LRG1), etc.) were found to create an ambiguous core involved in cancer development but also to exactly promote tumor progression in the early stages. Additionally, we identified post-translational modifications, which according to the literature are associated with the development of colorectal cancer, including kininogen 1 protein (T327-p), alpha-2-HS-glycoprotein (S138-p) and newly identified PTMs, i.e., vitamin D-binding protein (K75-ac and K370-ac) and plasma protease C1 inhibitor (Y294-p), which may also contribute and negatively impact on CRC progression. Conclusions: The contribution of cytokines and proteins of the extracellular matrix is the most significant factor in CRC development in the early stages. This can be concluded since tumor growth is tightly associated with chronic aseptic inflammation and concatenated malignancy related to loss of extracellular matrix stability. Due attention should be paid to Apolipoprotein E (APOE), Apolipoprotein C1 (APOC1), and Apolipoprotein B-100 (APOB) because of their impact on the malfunction of DNA repair and their capability to regulate mTOR and PI3K pathways. The contribution of the observed PTMs is still equivocal, but a significant decrease in the likelihood between modified and native proteins was not detected confidently.
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