Technology For Analysis Of Proteins Research Articles

Clinical practice guidelines for molecular tumor markers, 2nd edition review part 1.

With advances in gene and protein analysis technologies, many target molecules that may be useful in cancer diagnosis have been reported. Therefore, the "Tumor Marker Study Group" was established in 1981 with the aim of "discovering clinically" useful molecules. Later, the name was changed to "Japanese Society for Molecular Tumor Marker Research" in 2000 in response to the remarkable progress in gene-related research. Currently, the world of cancer treatment is shifting from the era of representative tumor markers of each cancer type used for tumor diagnosis and treatment evaluation to the study of companion markers for molecular-targeted therapeutics that target cancer cells. Therefore, the first edition of the Molecular Tumor Marker Guidelines, which summarizes tumor markers and companion markers in each cancer type, was published in 2016. After publication of the first edition, the gene panel testing using next-generation sequencing became available in Japan in June 2019 for insured patients. In addition, immune checkpoint inhibitors have been indicated for a wide range of cancer types. Therefore, the 2nd edition of the Molecular Tumor Marker Guidelines was published in September 2021 to address the need to revise the guidelines. Here, we present an English version of the review (Part 1) of the Molecular Tumor Marker Guidelines, Second Edition.

The Intriguing Landscape of Single-Cell Protein Analysis.

Profiling protein expression at single‐cell resolution is essential for fundamental biological research (such as cell differentiation and tumor microenvironmental examination) and clinical precision medicine where only a limited number of primary cells are permitted. With the recent advances in engineering, chemistry, and biology, single‐cell protein analysis methods are developed rapidly, which enable high‐throughput and multiplexed protein measurements in thousands of individual cells. In combination with single cell RNA sequencing and mass spectrometry, single‐cell multi‐omics analysis can simultaneously measure multiple modalities including mRNAs, proteins, and metabolites in single cells, and obtain a more comprehensive exploration of cellular signaling processes, such as DNA modifications, chromatin accessibility, protein abundance, and gene perturbation. Here, the recent progress and applications of single‐cell protein analysis technologies in the last decade are summarized. Current limitations, challenges, and possible future directions in this field are also discussed.

Single-cell analysis targeting the proteome.

The existence of cellular heterogeneity and its central relevance to biological phenomena provides a strong rationale for a need for analytical methods that enable analysis at the single-cell level. Analysis of the genome and transcriptome is possible at the single-cell level, but the comprehensive interrogation of the proteome with this level of resolution remains challenging. Single-cell protein analysis tools are advancing rapidly, however, and providing insights into collections of proteins with great relevance to cell and disease biology. Here, we review single-cell protein analysis technologies and assess their advantages and limitations. The emerging technologies presented have the potential to reveal new insights into tumour heterogeneity and therapeutic resistance, elucidate mechanisms of immune response and immunotherapy, and accelerate drug discovery.

Qualitative and quantitative proteomic analysis of formalin-fixed paraffin-embedded (FFPE) tissue.

Formalin-fixed, paraffin-embedded (FFPE) tissue has recently gained interest as an alternative to fresh/frozen tissue for retrospective protein biomarker discovery. However, during the formalin fixation proteins undergo degradation and cross-linking, making conventional protein analysis technologies challenging. Cross-linking is even more challenging when quantitative proteome analysis of FFPE tissue is planned. The use of conventional protein labeling technologies on FFPE tissue has turned out to be problematic as the lysine residue labeling targets are frequently blocked by the formalin treatment. We have established a qualitative and quantitative proteomics analysis technique for FFPE tissues that combines label-free proteomic analysis with optimized protein extraction and separation conditions.

The application of cell cultures, body fluids and tissues in oncoproteomics

Mass spectrometry (MS)-based proteomics is a rapidly developing technology for the large scale analysis of proteins, their interactions and subcellular localization. In recent years proteomics has attracted much attention in medicine. Since a single biomarker might not have sufficient sensitivity and specificity in clinical practice, the identification of biomarker panels that comprise several proteins would improve the detection and clinical management of cancer patients. Additionally, the characteristics of protein profiles of most severe human malignancies certainly contribute to the understanding of the biology of cancer and fill the gap in our knowledge of carcinogenesis. This knowledge also is likely to result in the discovery of novel potential cancer markers and targets for molecular therapeutics. It is believed that the novel biomarkers will help in the development of personalized therapy tailored to the individual patient and will thereby reduce the mortality rate from cancer. In this review, the use of different types of human clinical samples (cell cultures, tissues and body fluids) in oncoproteomics is explained and the latest advances in mass spectrometry-based proteomics biomarker discovery are discussed.

Rapid and deep proteomes by faster sequencing on a benchtop quadrupole ultra-high-field Orbitrap mass spectrometer.

Shotgun proteomics is a powerful technology for global analysis of proteins and their post-translational modifications. Here, we investigate the faster sequencing speed of the latest Q Exactive HF mass spectrometer, which features an ultra-high-field Orbitrap mass analyzer. Proteome coverage is evaluated by four different acquisition methods and benchmarked across three generations of Q Exactive instruments (ProteomeXchange data set PXD001305). We find the ultra-high-field Orbitrap mass analyzer to be capable of attaining a sequencing speed above 20 Hz, and it routinely exceeds 10 peptide spectrum matches per second or up to 600 new peptides sequenced per gradient minute. We identify 4400 proteins from 1 μg of HeLa digest using a 1 h gradient, which is an approximately 30% improvement compared to that with previous instrumentation. In addition, we show that very deep proteome coverage can be achieved in less than 24 h of analysis time by offline high-pH reversed-phase peptide fractionation, from which we identify more than 140,000 unique peptide sequences. This is comparable to state-of-the-art multiday, multienzyme efforts. Finally, the acquisition methods are evaluated for single-shot phosphoproteomics, where we identify 7600 unique HeLa phosphopeptides in one gradient hour and find the quality of fragmentation spectra to be more important than quantity for accurate site assignment.

Molecular characterisation of high molecular weight glutenin allele Glu-B1h encoding 1Bx14+1By15 subunits in bread wheat (Triticum aestivum L.)

In this study, the authentic high molecular weight glutenin (HMW-GS) allele Glu-B1 h encoding for subunits 1Bx14 and 1By15 from German bread wheat cultivars Hanno and Imbros was identified and cross-verified by a suite of established protein analysis technologies, including sodium dodecyl sulfate-polyacrylamide gel electrophoresis, reversed-phase high-performance liquid chromatography, reversed-phase ultra-performance liquid chromatography, and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS). The complete encoding sequences were isolated by allele-specific PCR, and consist of 2367 bp for 1Bx14 and 2151 bp for 1By15 and encode 789 and 717 amino acid residues, respectively. The deduced molecular masses of two subunit genes were 82 340.13 Da and 74 736.13 Da, corresponding well to those determined by MALDI-TOF-MS. The presence and authenticity of 1Bx14 and 1By15 subunits were further confirmed by liquid chromatography coupled to tandem mass spectrometry and heterologous expression in E. coli. Comparative analysis demonstrated that 1Bx14 possessed one deletion and 20 single-nucleotide polymorphism variations compared with seven other Glu-B1 x-type HMW-GS genes that mainly resulted from C–T substitutions, whereas compared with five other Glu-B1 y-type HMW-GS genes, 1By15 displayed few variations. Phylogenetic analysis based on the complete coding sequences of the published HMW-GS genes showed that 1Bx14 had a high divergence with other 1Bx subunit genes, whereas 1By15 displayed greater similarity with 1By20. A possible evolutionary route for 1Bx14 gene formation is proposed, which might have resulted from an intra-strand illegitimate recombination event that occurred ~1.32 million years ago.

Formalin-Fixed Paraffin-Embedded (FFPE) Proteome Analysis Using Gel-Free and Gel-Based Proteomics

Formalin-fixed paraffin-embedded (FFPE) tissue has recently gained interest as an alternative to fresh/frozen tissue for retrospective protein biomarker discovery. However, during the fixation process, proteins undergo degradation and cross-linking, making conventional protein analysis technologies problematic. In this study, we have compared several extraction and separation methods for the analysis of proteins in FFPE tissues. Incubation of tissue sections at high temperature with a novel extraction buffer (20 mM Tris-HCl, pH 8.8, 2% SDS, 1% beta-octylglucoside, 200 mM DTT, 200 mM glycine, and a mixture of protease inhibitors) resulted in improved protein recovery. Protein separation by 1-DE followed by LC-ESI MS/MS analysis was the most effective approach to identify proteins, based on the number of peptides reliably identified. Interestingly, a number of peptides were identified in regions of the 1DE not corresponding to their native molecular weights. This is an indication of the formation of protein-protein complexes by cross-linking, and of protein fragmentation due to prolonged sample storage. This study will facilitate the development of future proteomic analysis of FFPE tissue and provide a tool for the validation in archival samples of biomarkers of exposure, prognosis and disease.

BiPS, a Photocleavable, Isotopically Coded, Fluorescent Cross-linker for Structural Proteomics

Cross-linking combined with mass spectrometry is an emerging approach for studying protein structure and protein-protein interactions. However, unambiguous mass spectrometric identification of cross-linked peptides derived from proteolytically digested cross-linked proteins is still challenging. Here we describe the use of a novel cross-linker, bimane bisthiopropionic acid N-succinimidyl ester (BiPS), that overcomes many of the challenges associated with other cross-linking reagents. BiPS is distinguished from other cross-linkers by a unique combination of properties: it is photocleavable, fluorescent, homobifunctional, amine-reactive, and isotopically coded. As demonstrated with a model protein complex, RNase S, the fluorescent moiety of BiPS allows for sensitive and specific monitoring of the different cross-linking steps, including detection and isolation of cross-linked proteins by gel electrophoresis, determination of in-gel digestion completion, and fluorescence-based separation of cross-linked peptides by HPLC. The isotopic coding of BiPS results in characteristic ion signal "doublets" in mass spectra, thereby permitting ready detection of cross-linker-containing peptides. Under MALDI-MS conditions, partial photocleavage of the cross-linker occurs, releasing the cross-linked peptides. This allows differentiation between dead-end, intra-, and interpeptide cross-links based on losses of specific mass fragments. It also allows the use of the isotope doublets as mass spectrometric "signatures." A software program was developed that permits automatic cross-link identification and assignment of the cross-link type. Furthermore photocleavage of BiPS assists in cross-link identification by allowing separate tandem mass spectrometry sequencing of each peptide comprising the original cross-link. By combining the use of BiPS with MS, we have provided the first direct evidence for the docking site of a phosphorylated G-protein-coupled receptor C terminus on the multifunctional adaptor protein beta-arrestin, clearly demonstrating the broad potential and application of this novel cross-linker in structural and cellular biology.

Mathematical Models of Oversaturated Protein Spots

Two-dimensional electrophoresis is used for protein separation in a special gel according isoelectric point and molecular mass, and remains one of most progressive technologies for protein analysis. As a result, gels are digitized and their images are process with software. The dynamic diapason of intensities of protein spots is very wide, thus software tools become too sensitive to various artefacts inevitable present in gel images. Often matching with reference or modelling of protein spot intensity distribution is done willing to get better protein spot estimates. Nevertheless that many protein spot models exist, they take into account only particular spot shape artefact. In the article modern protein spot models applicable in two-dimensional electrophoresis and DNR arrays are analysed. It is shown that symmetry is preserved only for protein spots in DNR arrays. Three new protein spots shape models – 4G, 4S and Bell shape – are presented in order to cope with shape distortion and asymmetry. Results of experimentation with 84 distorted protein spots applying three developed shape models and one of most popular – Flat-Top shape model are presented. It is shown that Bell shape model fits best to oversaturated protein spots while 4G shape model fits best to non-saturated spots and requires smallest computational time. Ill. 5, bibl. 11 (in English; summaries in English, Russian and Lithuanian).

The future of proteomic analysis in biological systems and molecular medicine

Proteomics is the study of proteins and their interactions within complex biological systems. While this field is often associated with mass spectrometry, it is more useful to consider proteomics in the context of an objective: to identify and understand the molecular basis of health and disease at the protein level in vivo. Achieving this objective will require (1) technological developments to resolve current instrument limitations and (2) multidisciplinary integration of biological and protein analysis technologies to answer important questions in both the biological sciences and molecular medicine.

Protein ligation: an enabling technology for the biophysical analysis of proteins

Biophysical techniques such as fluorescence spectroscopy and nuclear magnetic resonance (NMR) spectroscopy provide a window into the inner workings of proteins. These approaches make use of probes that can either be naturally present within the protein or introduced through a labeling procedure. In general, the more control one has over the type, location and number of probes in a protein, then the more information one can extract from a given biophysical analysis. Recently, two related approaches have emerged that allow proteins to be labeled with a broad range of physical probes. Expressed protein ligation (EPL) and protein trans-splicing (PTS) are both intein-based approaches that permit the assembly of a protein from smaller synthetic and/or recombinant pieces. Here we provide some guidelines for the use of EPL and PTS, and highlight how the dovetailing of these new protein chemistry methods with standard biophysical techniques has improved our ability to interrogate protein function, structure and folding.

Nanoscale Glassification of Gold Substrates for Surface Plasmon Resonance Analysis of Protein Toxins with Supported Lipid Membranes

Surface plasmon resonance (SPR) spectroscopy, a powerful tool for biosensing and protein interaction analysis, is currently confined to gold substrates and the relevant surface chemistries involving dextran and functional thiols. Drawbacks of using self-assembled monolayers (SAMs) for SPR-related surface modification include limited stability, pinhole defects, bioincompatibility, and nonspecific protein adsorption. Here we report the development of stable nanometer-scale glass (silicate) layers on gold substrates for SPR analysis of protein toxins. The nanoscale silicate layers were built up with layer-by-layer deposition of poly(allylamine hydrochloride) and sodium silicate, followed by calcination at high temperature. The resulting silicate films have a thickness ranging from 2 to 15 nm and demonstrate outstanding stability in flow cell conditions. The use of these surfaces as a platform to construct supported bilayer membranes (SBMs) is demonstrated, and improved performance against protein adsorption on SBM-coated surfaces is quantified by SPR measurements. SBMs can be formed reproducibly on the silicate surface via vesicle fusion and quantitatively removed using injection of 5% Triton X-100 solution, generating a fresh surface for each test. Membrane properties such as lateral diffusion of the SBMs on the silicate films are characterized with photobleaching methods. Studies of protein binding with biotin/avidin and ganglioside/cholera toxin systems show detection limits lower than 1 microg/mL (i.e., nanomolar range), and the response reproducibility is better than 7% RSD. The method reported here allows many assay techniques developed for glass surfaces to be transferred to label-free SPR analysis without the need for adaptation of protocols and time-consuming synthetic development of thiol-based materials and opens new avenues for developing novel bioanalytical technologies for protein analysis.

Proteomic studies in plants.

Proteomics is a leading technology for the high-throughput analysis of proteins on a genome-wide scale. With the completion of genome sequencing projects and the development of analytical methods for protein characterization, proteomics has become a major field of functional genomics. The initial objective of proteomics was the large-scale identification of all protein species in a cell or tissue. The applications are currently being extended to analyze various functional aspects of proteins such as post-translational modifications, protein-protein interactions, activities and structures. Whereas the proteomics research is quite advanced in animals and yeast as well as Escherichia coli, plant proteomics is only at the initial phase. Major studies of plant proteomics have been reported on subcellular proteomes and protein complexes (e.g. proteins in the plasma membranes, chloroplasts, mitochondria and nuclei). Here several plant proteomics studies will be presented, followed by a recent work using multidimensional protein identification technology (MudPIT).