Understanding the fundamentals of the on-off retention mechanism of oligonucleotides and their application to high throughput analysis

<p><strong>Our Library never stops expanding. What are the most recent contributions to LabRulezLCMS Library in the week of 13th January 2025? Check out new documents from the field of liquid phase, especially HPLC and LC/MS techniques!</strong></p><blockquote><p><span style="font-size:20px;">👉 </span><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library"><span style="font-size:20px;"><strong>SEARCH THE LARGEST REPOSITORY OF DOCUMENTS ABOUT LCMS AND RELATED TECHNIQUES</strong></span></a></p></blockquote><blockquote><p><strong>👉 Need info about different analytical techniques?</strong> Peek into <a target="_blank" rel="noopener noreferrer" href="https://gcms.labrulez.com/library"><strong>LabRulezGCMS</strong></a> or <a target="_blank" rel="noopener noreferrer" href="https://icpms.labrulez.com/library"><strong>LabRulezICPMS</strong></a> libraries.</p></blockquote><p>This week we bring you applications by <strong>Thermo Fisher Scientific,</strong> <strong>Shimadzu</strong>,<strong> </strong>a presentation by <strong>Agilent Technologies</strong>, a manual by <strong>Metrohm,</strong> and a poster from <strong>ASMS </strong>by <strong>Waters Corporation</strong>!</p><h4><strong>1. Thermo Fisher Scientific: High-resolution DIA proteomics workflow for single-cell samples on the Orbitrap Astral mass spectrometer</strong></h4><ul><li>Technical note</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/paper/28638"><strong>Full PDF for download</strong></a></li></ul><p>Assess proteome coverage and sample throughput performance for single-cell samples using a library-free and library-based data-independent acquisition (DIA) method on the Thermo Scientific™ Orbitrap™ Astral™ mass spectrometer.</p><h5><strong>Introduction</strong></h5><p>In recent years, there has been a shift to analyzing smaller sample quantities, driven by the interest in profiling the proteome of individual cells. This increased focus on LC-MS analysis of limited sample amounts, e.g., single cells, requires the highest possible sensitivity. At the same time, throughput and chromatographic performance must be preserved to generate sufficiently high-quality data from large data sets to draw meaningful conclusions. This trend toward analyzing smaller sample quantities reflects the need to understand the heterogeneous nature of biology through the dissection of complex systems into their individual parts or, specifically, individual cells. Traditional proteomics approaches read out the average of a sample while single-cell proteomics provides insights into the exact nature of each of the components in such samples.</p><p>The Orbitrap Astral mass spectrometer delivers a new standard for single-cell samples with deeper proteome coverage and faster throughput. It enables these capabilities through the synchronized acquisition of high-resolution and high-dynamic range full scan spectra with the Thermo Scientific™ Orbitrap™ mass analyzer and high speed and sensitivity MS/MS spectra with the Thermo Scientific™ Astral™ mass analyzer. The Thermo Scientific™ Vanquish™ Neo UHPLC system delivers maximum performance in terms of precise, low flow rates and gradient formation, as well as low injection volumes—an important benefit for single-cell proteomics and limited sample amounts.</p><p>The Thermo Scientific™ μPAC™ Neo HPLC columns with novel micro-pillar array deliver highly reproducible chromatographic separation and increased sensitivity with maximum resolution.<br>The Thermo Scientific™ FAIMS Pro Duo interface enhances sensitivity by removing background signals.</p><p>In this technical note, we present a single-cell proteomics workflow (Figure 1). To first evaluate sensitivity and quantitative precision and accuracy and linearity of the workflow, a dilution series from 5 ng to 50 pg of the Thermo Scientific™ Pierce™ HeLa protein digest standard was analyzed.</p><h4><strong>2. Shimadzu: Automatic Optimization of Gradient Conditions by AI Algorithm - Consecutive Optimization at Different Column Oven Temperatures</strong></h4><ul><li>Technical note</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.cz/paper/28642"><strong>Full PDF for download</strong></a></li></ul><h5><strong>User Benefits</strong></h5><ul><li>The AI algorithm of LabSolutions MD can automatically optimize gradient conditions to greatly reduce labor of LC method development.</li><li>Gradient conditions are automatically optimized at different column oven temperatures consecutively.</li><li>Automatic optimization of gradient conditions can be applied not only to new method development, but also to existing method to efficiently improve resolution.</li></ul><h5><strong>Introduction</strong></h5><p>In the typical LC method development, the process begins with “preparation” which includes mobile phase preparation, column installation, and creation of analysis schedules, then the analysis is started. After that, the acquired data is analyzed and “preparation” for the subsequent analysis is carried out, followed by starting the next analysis again. The method development progresses by repeating these processes, but in addition to the significant time required to repeatedly create analysis schedules, expertise in chromatography is necessary to explore optimal conditions based on data analysis. In other words, typical method development requires “human intervention”. Therefore, eliminating human involvement and automating such method development processes would be desirable to improve labor efficiency. This article introduces an approach for automatically optimizing gradient conditions at different column oven temperatures consecutively, identifying the combination of temperature and gradient conditions that meet the resolution criteria.</p><h5><strong>Conclusion</strong></h5><p>Automatic optimization of gradient conditions using AI algorithm of LabSolutions MD was applied to a model sample (mixture of seven compounds of small molecule) at different column oven temperatures. As a result, the temperature and gradient conditions that met the resolution criteria were successfully explored. In method development, human intervention, such as analysis batch creation and data analysis, is required to optimize gradient conditions. LabSolutions MD can provide significant labor savings in this area. For more information on LabSolutions MD, please refer to the Technical Report <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/c190_e284_1ca86b48ea/c190-e284.pdf">“Efficient Method Development Based on Analytical Quality by Design with LabSolutions MD Software (C190-E284)”</a>.</p><h4><strong>3. Agilent Technologies: Optimize Your Polymer Analysis by Selecting the Ideal GPC/SEC Column</strong></h4><ul><li>Presentation</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/paper/28578"><strong>Full PDF for download</strong></a></li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/4481"><strong>Recorded webinar</strong></a></li></ul><p>&nbsp;</p><ul><li><strong>GPC/SEC Separation Mechanism</strong></li><li><strong>Conventional GPC/SEC Workflow</strong></li><li><strong>Considerations for GPC/SEC Column Selection (Key questions to ask)</strong><ul><li>What polymer are you analysing?</li><li>Which solvent (or solvents) is your polymer soluble in?</li><li>What is the expected molecular weight range of your polymer?</li><li>What is the requirement for your analysis or what would you like to improve about your existing GPC/SEC separation?<ul><li>Resolution is important</li><li>Reproducibility of sample chromatography and results</li><li>Speed of analysis or sample throughput is something to improve on</li></ul></li></ul></li><li><strong>Further considerations</strong><ul><li>Know the properties of the sample</li><li>Be familiar with the properties of the columns being considered</li><li>It is important to balance polarities for the sample, solvent, and column packing</li></ul></li><li><strong>Common Column Chemistries for GPC/SEC</strong></li><li><strong>Agilent Magic Triangles</strong></li><li><strong>Column Selection: Porosity</strong></li><li><strong>Individual Pore Versus MultiPorous Particle</strong></li><li><strong>Column Selection: Particle size</strong></li><li><strong>Columns for Low Polarity Organic Eluents</strong></li><li><strong>Columns for Medium Polarity Organic Eluents</strong></li><li><strong>Columns for High Temperature GPC/SEC</strong></li><li><strong>Columns for Fluorinated Organic Eluents</strong></li><li><strong>Columns for Aqueous Eluents: Neutral or weak Anions</strong></li><li><strong>Columns for Aqueous Eluents: Polycations and Strong Polyanions</strong></li><li><strong>Column Selection: Additional Considerations</strong></li><li><strong>Columns in Series</strong></li><li><strong>Wrongly Coupled Columns</strong></li><li><strong>Combination of Individual Pore Size Columns</strong></li><li><strong>Column Selection and Importance of Solvent Choice</strong></li><li><strong>Solvent Selection</strong></li><li><strong>Successful Solvent Choice</strong></li><li><strong>Eluent Modification in Organic GPC</strong></li><li><strong>Common Questions Around GPC/SEC Standards</strong></li><li><strong>Agilent Polymer Calibration Standard Offerings</strong></li><li><strong>Well Characterized Polymer Standards</strong></li><li><strong>Easivial Certificate of Analysis</strong></li><li><strong>Common Questions Around SEC Standards</strong></li><li><strong>Installation and Care of Your Column</strong></li><li><strong>Before Your Analysis</strong></li><li><strong>Column Selection – Additional considerations</strong></li><li><strong>System Tubing</strong></li><li><strong>Your GPC/SEC Toolbox</strong></li></ul><h4><strong>4. Metrohm: Installation Instruction for ProfIC Vario 7 Cation</strong></h4><ul><li>Manual</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.cz/paper/28584"><strong>Full PDF for download</strong></a></li></ul><p>The ProfIC Vario 7 Cation is a Professional IC Vario system with Inline Dilution and Inline Dialysis for the fully automatic determination of cations in high concentrations in matrices containing emulsions. This system enables users to dilute the sample and eliminate droplets of fat or organic solvents or particles prior to the analysis.</p><h4><strong>5. Waters Corporation: IMPROVED TISSUE HOMOGENIZATION AND SPE-BASED SAMPLE PREPARATIONS FOR THE QUANTITATIVE LC-MS ANALYSIS OF OLIGONUCLEOTIDE THERAPEUTICS</strong></h4><ul><li>Poster/ASMS</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/paper/27580"><strong>Full PDF for download</strong></a></li></ul><p>Efficient extraction of drug analytes is a critical aspect of drug metabolism and pharmacokinetics (DMPK) studies. This has long applied to small molecules and still applies to the bioanalysis of oligonucleotides. Oligonucleotide drugs and their metabolites must be quantified within both biofluids and tissue samples. The newest of oligonucleotide drugs are both extensively modified and conjugated. These modified residues and conjugate moieties can complicate extraction recovery and reproducibility. In this work, we report several key insights on how to achieve improved extractions. Protocols for solvent-assisted proteinase K sample pre-treatments are investigated using a weak anion exchange (WAX) microplate-based solid phase extraction (SPE) device. Direct injection LC-MS quantitation is demonstrated for all three antisense oligonucleotides (ASOs).</p><h5><strong>CONCLUSIONS</strong></h5><ul><li>Selective extraction and elution of ASO oligonucleotides is feasible from multiple tissue types.</li><li>Extracted ASOs are amenable for either full scan mode analysis on benchtop small footprint TOF MS or MRM analysis by Triple Quad MS</li><li>Reproducible and repeatable data were collected even without the application of an internal standard</li><li>One novel aspect of the SPE eluate is that no further sample preparation is required for subsequent LC-MS analysis saving time and avoiding sample losses during drying and reconstitution</li><li>Eluted nucleic acid components can be readily analyzed by regular ion-pairing reversed phase chromatography and UV/MS-based detection and quantitation</li><li>This robust tissue extraction protocols for improved LC-MS based bioanalysis of ASO drugs was repeatable and highly reproducible across multiple batches of SPE sorbent, providing confidence in the suitability of the optimized protocols</li></ul>

News from LabRulezLCMS Library - Week 3, 2025

<p><strong>In the week of January 20, the following webinars await you in the field of liquid phase and especially HPLC and LC/MS.</strong></p><blockquote><p><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinars"><strong>👉 DETAILS AND REGISTRATION IN THE WEBINARS SECTION</strong></a></p><p><strong>👉 Search and filter in the largest global database</strong> with over&nbsp;</p><p><strong>4 000 GC, LC, MS and spectroscopy webinars</strong>.</p></blockquote><h5><strong>1. Thermo Fisher Scientific: Characterization and sequence mapping of large RNA and mRNA therapeutics using HRAM</strong></h5><div><div><ul><li><div>Tu, 21.1.2025 11:00 CET</div></li><li><div><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/4580">Registration</a></div></li></ul></div><p>The presentation showcases the innovative partial T1 digest strategy developed at the University of Sheffield for sequencing mRNA therapeutics.</p></div><h5><strong>2.&nbsp;SelectScience/Agilent Technologies: From Start to Finish: How Agilent Can Support Better Decisions in IVT mRNA-Based Biopharmaceutical Analysis</strong></h5><div><div><ul><li><div>Tu, 21.1.2025 17:00 CET</div></li><li><div><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/4674">Registration</a></div></li></ul></div><p>We will discuss how automated electrophoresis and LC/MS systems solutions from Agilent provide reliable insights for IVT mRNA workflows at many steps.</p></div><h5><strong>3.&nbsp;Phenomenex: LC Troubleshooting Quick Guide: Spot Problems Fast &amp; Fix Them Like a Pro!</strong></h5><div><div><ul><li><div>Th, 23.1.2025 11:00 CET</div></li><li><div><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/4676">Registration</a></div></li></ul></div><p>In this webinar - General troubleshooting approach, HPLC troubleshooting steps and diagnosing tips and preventive maintenance and column care.</p></div><h5><strong>4.&nbsp;Thermo Fisher Scientific: State-of-the-art E&amp;L is not rocket science</strong></h5><div><div><ul><li><div>Th, 23.1.2025 11:00 CET</div></li><li><div><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/4581">Registration</a></div></li></ul></div><p>Ulli Honester, a team leader from Solvias, presented on advancements in mass spectrometry for identifying extractables and leachables (E&amp;L).</p></div><h5><strong>5.&nbsp;Waters Corporation: Empower Tips: Ask Me Anything About Navigating Data Governance - Risk and Vigilant Monitoring</strong></h5><div><div><ul><li><div>Th, 23.1.2025 16:00 CET</div></li><li><div><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/4550">Registration</a></div></li></ul></div><p>We will explore critical aspects of data governance and critical thinking, insights and strategies to help navigate the current environment, evolving your approach and modernizing your way of working.</p></div><h5><strong>6.&nbsp;Evosep Biosystems: A beginner’s guide to Evosep 2025</strong></h5><div><div><ul><li><div>Th, 23.1.2025 16:00 CET</div></li><li><div><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/4548">Registration</a></div></li></ul></div><p>This webinar is your opportunity to gain a comprehensive understanding of the Evosep One’s key features and how it can seamlessly integrate into your current processes.</p></div><h5><strong>7.&nbsp;Shimadzu Corporation: ASTM D8421 and D8535: Cost Effective Methods for Rapid PFAS Testing in Wastewater and Soil</strong></h5><div><div><ul><li><div>Th, 23.1.2025 20:00 CET</div></li><li><div><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/4465">Registration</a></div></li></ul></div><p>Discover simplified standardized methods for PFAS analysis based on co-solvation as sample preparation.</p></div>

Webinars LabRulezLCMS Week 4/2025

Article

Webinars

Application

Scientific article

Various

Everything from the world of analytical chemistry in one place. We connect people in solving their problems. At Labrulez you will find all the necessary information easily, quickly and clearly. Stop searching and start finding.

LabRulez

Quantitative analysis of proteins (Bioanalytic) 

In our laboratory, proteomic analyses, including quantitative analysis of proteins, are performed by HPLC-MS or HPLC-MS/MS methods on a ZenoTOF 7600 high-resolution mass spectrometer (SCIEX).

<p><strong>Quantitative analysis of proteins is important in many industries and applications. Proteins include a diverse group of compounds that participate in virtually every biological process. Proteins are building blocks, hormones, transport molecules, cell receptors, cell growth and differentiation factors. Because of this, the analysis of protein interactions is crucial in drug design and production.</strong></p><p>Proteins are macromolecules - the basis of their structure are polypeptide chains containing from 100 to more than 10000 amino acids (10 - 100 kDa). Proteins are found in simple and complex forms. Complex proteins contain a non-protein component (sugar, lipids, metal ions or dyes) in their structure.</p><p>In our laboratory, proteomic analyses, including quantitative analysis of proteins, are performed by HPLC-MS or HPLC-MS/MS methods on a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?manufacturerItems=52&amp;search=ZenoTOF+7600"><strong>ZenoTOF 7600</strong></a> high-resolution mass spectrometer (SCIEX). We process the obtained results using Sciex OS, Spectronaut, Skyline software.</p><h4><strong>MS vs MS/MS</strong></h4><p>Quantitative analysis of intact protein (whole protein in intact form) leads to the concentration (determined from a prepared curve from a standard (with or without matrix) and confirmation of the identification by superimposing (on a mirrored basis) the MS spectrum of the protein determined in the sample to the MS spectrum of the standard. For your needs, we can generate a list of protein-structured compounds (impurities) that were identified in the sample and determine the ratio of their signal area to the signal area of the analyzed protein.</p><p>The result of proteomic quantitative peptide analysis allows precise and much more sensitive determination of protein concentration. The analysis is based on MS/MS data obtained for peptides obtained by digestion with proteolytic enzymes. The reported result consists of the concentration of the protein under study and confirmation of its presence based on the determined amino acid sequence. For such analyses, it is recommended to add standards internal standards.</p><blockquote><p><span style="font-size:22px;"><strong>Our offer:</strong></span></p><ul><li>For proteins and peptides with low masses - intact analysis with MS detection;</li><li>Proteins with masses &gt;20kDa require optimization of the digestion method and MS/MS detection. This results in specific signals from the analyzed protein in the mixture/matrix;</li><li>From complex matrices (including serum, cell culture, fungal suspension), sample analysis involves several steps:&nbsp;<ul><li>Sample purification;</li><li>Digestion to obtain unique peptides to distinguish the analyzed proteins from other proteins in the sample;</li><li>Sample preparation requires appropriate method optimization and validation (let's set validation criteria individually);</li></ul></li><li>Reported quantitative result includes confirmation of the presence of the protein in native form or a peptide derived from it (usually confirmation includes at least two peptide sequences);</li><li>Statistical analysis using tools: MarkerView (PCA), GraphPad Prism, Trends, Sciex OS.</li></ul><p><i>To optimize and validate the method, it is necessary to provide a standard, in the case of proteins with high masses - it is recommended to add an internal standard.</i></p><p>See also:</p><ul><li><a target="_blank" rel="noopener noreferrer" href="https://labexperts.com.pl/service/identyfikacja-bialek/"><strong>Protein identification</strong></a><strong>,</strong></li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1296"><strong>Analysis and characterization of monoclonal antibodies</strong></a><strong>.</strong></li></ul></blockquote><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Bioanalytic_Bioanalytic_Quantitative_analysis_of_proteins_1_de494646f2.jpg" alt="Bioanalytic: Quantitative analysis of proteins." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Bioanalytic_Bioanalytic_Quantitative_analysis_of_proteins_1_de494646f2.jpg 200w," sizes="100vw" width="777" height="606"></p>

Identification of the substance – what is in your sample? (Bioanalytic)

We are able to identify metabolites and products of chemical reactions, contaminants of biological and chemical substances and preparations, and perform profiling of a series of samples to study the kinetics of transformations.

<h4><strong>Substance Identification - Looking for answers to what's in your sample? - we can help you.</strong></h4><p>Substance identification based on LC-MS/MS techniques can involve single compounds or mixtures of substances. In our analyses, we rely primarily on the knowledge and skills we have acquired after many years of work. For analysis, we use full laboratory facilities for sample preparation and high-resolution mass spectrometry (<a target="_blank" rel="noopener noreferrer" href="https://labexperts.com.pl/o-nas/">equipment</a>).</p><p>We use different work algorithms depending on the material we are dealing with and the analytical task to be solved. We use DDA (IDA) or DIA (SWATH) methods or targeted approaches (e.g. MRMhr) if we know what to expect. Thus, we are able to identify metabolites and products of chemical reactions, contaminants of biological and chemical substances and preparations, and perform profiling of a series of samples to study the kinetics of transformations.</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Bioanalytic_Bioanalytic_Identification_of_the_substance_what_is_in_your_sample_2_0f425a648b.jpg" alt="Bioanalytic: Identification of the substance – what is in your sample" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Bioanalytic_Bioanalytic_Identification_of_the_substance_what_is_in_your_sample_2_0f425a648b.jpg 245w," sizes="100vw" width="1881" height="1156"></p><h4><strong>COST OF SERVICE</strong></h4><p>We determine the cost of the service individually. The final cost is influenced by many factors related to the nature of the sample itself and the client's expectations. Thus, the price depends, among other things, on the complexity of the sample preparation procedure, the scope and number of analyses, etc.</p><p><strong>The service includes (depending on individual arrangements):</strong></p><h5><strong>Development of sample preparation procedure</strong></h5><ul><li>Depending on the matrix and test substances.</li></ul><h5><strong>Development of analytical methods</strong></h5><ul><li>Application of high-resolution LC-MS/MS analyses using DDA (IDA) or DIA (SWATH) experiments and software to support the development of targeted screening methods.</li></ul><h5><strong>Analysis of results includes:</strong></h5><ul><li>finding and identifying the substances of interest, interpreting MS/MS spectra and proposing a structural formula.</li><li>Comparative analysis of sample series, e.g. PCA,</li><li>profiling of analytes in sample series</li></ul><h5><strong>Reporting of results</strong></h5><ul><li>Compile results into easy-to-read reports.</li></ul><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Bioanalytic_Bioanalytic_Identification_of_the_substance_what_is_in_your_sample_3_c1b8adc1d0.jpg" alt="Bioanalytic: Identification of the substance – what is in your sample" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Bioanalytic_Bioanalytic_Identification_of_the_substance_what_is_in_your_sample_3_c1b8adc1d0.jpg 245w," sizes="100vw" width="1919" height="1208"></p>

Characteristics of monoclonal antibodies (Bioanalytic)

At LabExperts, proteomic analyses are performed on a ZenoTOF 7600 high-resolution LC-MS/MS system (SCIEX). For MS/MS analyses, we use Collision induced dissociation (CID) and Electron-Activated Dissociation (EAD) fragmentation.

<p><strong>Antibodies are molecules made up of 4 polypeptide chains: two heavy (H - high-chain) linked to each other by a specific type of chemical bond - a disulfide bridge, and two light (L - light-chain) linked by an identical bond to the heavy chains. Monoclonal antibodies (mABs) are used in various forms - as free molecules, in the form of a drug-antibody combination, and in the form of an immunoconjugate, a drug-antibody-biopolymer combination.</strong></p><p>Their main use is in highly specific targeted therapy for cancer, cardiovascular, autoimmune and nervous system diseases. They are also used in the treatment of osteoporosis, Alzheimer's disease or transplantation. The range of applications of monoclonal antibodies is systematically expanded. The introduction of a new antibody to the market requires complex product characterization, which involves numerous proteomic analyses, which are performed by LC-MS/MS techniques, among others.</p><h4><strong>LC-MS/MS</strong></h4><p>The characterization of monoclonal antibodies involves many aspects, including, but not limited to, a suite of analyses related to confirmation of the structure, chain sequence and degree of modification of the antibody. Post-translational modifications (PTMs) are chemical changes that occur after the translation process. These changes generally result in an increase in the immunogenicity of the drug. For this reason, it is essential to monitor modifications as critical quality attributes (CQAs) during both the production and storage of the drug. To ensure monitoring of CQA points, the primary structure of the protein is characterized based on peptide mapping (peptide mapping) analysis. The protein under study is subjected to proteolytic digestion using enzymes such as trypsin, chymotrypsin, Lys-C, Glu-C. As a result of various proteolytic enzymes, a wide range of peptides is obtained. Ultimately, coverage of the entire sequence of the protein under study is obtained.</p><figure class="image"><img style="aspect-ratio:1536/947;" src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Bioanalytic_Bioanalytic_Characteristics_of_monoclonal_antibodies_3_d94fa1623b.jpg" alt="Bioanalytic: Characteristics of monoclonal antibodies." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Bioanalytic_Bioanalytic_Characteristics_of_monoclonal_antibodies_3_d94fa1623b.jpg 245w," sizes="100vw" width="1536" height="947"></figure><p>At LabExperts, proteomic analyses are performed on a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?manufacturerItems=52&amp;search=ZenoTOF+7600"><strong>ZenoTOF 7600</strong></a> high-resolution LC-MS/MS system (SCIEX). For MS/MS analyses, we use Collision induced dissociation (CID) and Electron-Activated Dissociation (EAD) fragmentation. This provides us with data for unambiguous identification of protein-building amino acids. We process the results using Sciex OS, Skyline and Spectronaut software.</p><blockquote><p><span style="font-size:22px;"><strong>We perform:</strong></span></p><ul><li>Whole protein analyses - Intact - to determine the exact mass and confirm the theoretical mass;</li><li>N-glycosylation profile determination (based on MS spectrum reconstruction):<ul><li>Glycosylated form;</li><li>Deglycosylated;</li><li>Glycosylated with reduced disulfide bridges;</li><li>Deglycosylated with reduced disulfide bridges.</li></ul></li><li>Peptide maps:<ul><li>Protein digestion with various proteases so that sequence coverage is close to 100%<ul><li>Analysis of PTMs:</li><li>Location of modifications</li></ul></li><li>[%] of modifications compared to the unmodified sequence</li></ul></li><li>TiPOS testing - (technical proof of similarity) - technical confirmation of the similarity of the protein synthesized by the customer (from several LOTs) with the target protein (provided as a template).</li></ul><p>In order to maintain the highest possible analytical standards, we perform monoclonal antibody analyses under the regime of an internally developed and implemented laboratory quality system based on GLP principles.</p><p>See also our other proteomics services: <a target="_blank" rel="noopener noreferrer" href="https://labexperts.com.pl/service/identyfikacja-bialek/"><strong>Protein identification</strong></a>, <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1294"><strong>Protein quantitative analysis</strong></a></p></blockquote><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Bioanalytic_Bioanalytic_Characteristics_of_monoclonal_antibodies_2_cb93143fe6.jpg" alt="Bioanalytic: Bioanalytic Characteristics of monoclonal antibodies." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Bioanalytic_Bioanalytic_Characteristics_of_monoclonal_antibodies_2_cb93143fe6.jpg 245w," sizes="100vw" width="1536" height="908"></p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Bioanalytic_Bioanalytic_Characteristics_of_monoclonal_antibodies_1_0e8ab7cdfa.jpg" alt="Bioanalytic: Bioanalytic Characteristics of monoclonal antibodies." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Bioanalytic_Bioanalytic_Characteristics_of_monoclonal_antibodies_1_0e8ab7cdfa.jpg 245w," sizes="100vw" width="1536" height="858"></p>

Analysis of Anabolic steroids (Bioanalytic)

We offer a highly specific and sensitive qualitative analysis of a wide profile of anabolics by LC-HR-MS/MS technique (detection limits range from 0.01 to 5 ng/ml, depending on the substance).

<h4><strong>Anabolic substances in urine</strong></h4><p>The effect of anabolic steroids (<a target="_blank" rel="noopener noreferrer" href="https://en.wikipedia.org/wiki/Anabolic_steroid"><strong>more information</strong></a>) is to stimulate the growth of muscle mass and sometimes also bone mass by stimulating protein synthesis or altering calcium metabolism. This effect is often used in sports doping. However, anabolic steroids also have many side effects, as they strongly affect the body's overall hormonal balance.</p><p>Anabolic steroids used by purely recreational athletes are still not banned in most countries, including Poland. Nevertheless, their amateur use during adolescence often results in the inability to continue a professional sports career into adulthood or leads to hormonal imbalances in the body.</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Bioanalytic_Analysis_of_Anabolic_steroids_Bioanalytic_1_46fa2f41ca.jpg" alt="Bioanalytic: Analysis of Anabolic steroids." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Bioanalytic_Analysis_of_Anabolic_steroids_Bioanalytic_1_46fa2f41ca.jpg 245w," sizes="100vw" width="1200" height="740"></p><p>If you want to know whether and which anabolic substances are present in the samples of athletes, or you want to exclude doping in this area - use the offer carried out by us together with the company <a target="_blank" rel="noopener noreferrer" href="http://www.lab4tox.pl/"><strong>Lab4Tox</strong></a>. We offer a highly specific and sensitive qualitative analysis of a wide profile of anabolics by LC-HR-MS/MS technique (detection limits range from 0.01 to 5 ng/ml, depending on the substance). The use of high-resolution mass spectrometry (HR-MS and HR-MS/MS) in the analysis of anabolic steroids makes it possible to unequivocally confirm or exclude the presence of a given compound. The list of compounds at present includes:</p><ul><li><span class="ts-list-li-content">Boldenone undecylenate</span></li><li><span class="ts-list-li-content">Turinabol</span></li><li><span class="ts-list-li-content">Drostanolone enanthate</span></li><li><span class="ts-list-li-content">Drostanolone propionate</span></li><li><span class="ts-list-li-content">Methandienone</span></li><li><span class="ts-list-li-content">Methenolone acetate</span></li><li><span class="ts-list-li-content">Methenolone enanthate</span></li><li><span class="ts-list-li-content">Nandrolone phenylpropionate</span></li><li><span class="ts-list-li-content">Oxandrolone</span></li><li><span class="ts-list-li-content">Oxymetholone</span></li><li><span class="ts-list-li-content">Stanozolol</span></li><li><span class="ts-list-li-content">5-alpha-DHT</span></li><li><span class="ts-list-li-content">6-beta-hydroxymethandienone</span></li><li><span class="ts-list-li-content">19-norandrosterone</span></li><li><span class="ts-list-li-content">3'-hydroxystanazolol</span></li><li><span class="ts-list-li-content">4-beta-hydrozystanazolol</span></li><li><span class="ts-list-li-content">17-alpha-trenbolone</span></li><li><span class="ts-list-li-content">Testosterone caproate</span></li><li><span class="ts-list-li-content">Testosterone cypionate</span></li><li><span class="ts-list-li-content">Testosterone decanoate</span></li><li><span class="ts-list-li-content">Testosterone enanthate</span></li><li><span class="ts-list-li-content">Testosterone isocaproate</span></li><li><span class="ts-list-li-content">Testosterone phenylpropionate</span></li><li><span class="ts-list-li-content">Testosterone propionate</span></li><li><span class="ts-list-li-content">Testosterone undecanoate</span></li><li><span class="ts-list-li-content">Trenbolone acetate</span></li><li><span class="ts-list-li-content">Testosterone</span></li><li><span class="ts-list-li-content">Epitestosterone</span></li></ul><p><span class="ts-list-li-content">The list of compounds is not complete and will be systematically expanded according to customer needs.</span></p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Bioanalytic_Analysis_of_Anabolic_steroids_Bioanalytic_2_78729119b0.jpg" alt="Bioanalytic: Analysis of Anabolic steroids is carried out by us together with the company Lab4Tox." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Bioanalytic_Analysis_of_Anabolic_steroids_Bioanalytic_2_78729119b0.jpg 245w," sizes="100vw" width="625" height="100"></p><p><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products?category=54&amp;distributorItems=315"><span class="ts-list-li-content"><strong>Other analytical methods we offer</strong></span></a><span class="ts-list-li-content">.</span></p>

Identification of the proteins (Bioanalytic)

We perform proteomic analyses on a state-of-the-art high-resolution ZenoTOF 7600 (SCIEX), in which the Zeno ion trap and EAD (Electron-Activated Dissociation) technology together make a powerful combination.

<p><strong>Protein identification is based on experimental measurement of peptide molecular weights and matching the amino acid sequence of the protein under study with protein sequences deposited in libraries (databases). Two approaches are most commonly used in proteomic studies: top-down and bottom-up. The top-down method involves analyzing full protein sequences. Bottom-up analyses, on the other hand, use digestion of proteins with proteolytic enzymes. The peptides obtained by digestion are analyzed using a mass spectrometer. Each analysis leads to a comparison of experimental data with data obtained by theoretical digestion of proteins described in the database.</strong></p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Bioanalytic_Identification_of_the_proteins_Bioanalytic_2_6231b73f0d.jpg" alt="Bioanalytic: Identification of the proteins." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Bioanalytic_Identification_of_the_proteins_Bioanalytic_2_6231b73f0d.jpg 245w," sizes="100vw" width="1536" height="766"></p><h4><strong>LC-MS/MS</strong></h4><p>Tandem mass spectrometers are commonly used to determine protein sequences. They allow ion selection of pseudomolecular peptides and then fragmentation and measurement of the masses of the resulting fragments. Fragmentation using <i>collision induced dissociation</i>(CID ) is an invaluable tool in targeted and untargeted MS/MS analysis of small molecules. It is important to note that in proteomic analysis, data obtained using the aforementioned fragmentation method may leave gaps. For example, spectra obtained by CID fragmentation do not allow unambiguous differentiation of some amino acids: leucine from isoleucine (identical molar masses and CID fragmentation) and lysine from glutamine (very similar masses). For this reason, we perform proteomic analyses on a state-of-the-art high-resolution <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?manufacturerItems=52&amp;search=+ZenoTOF+7600"><strong>ZenoTOF 7600</strong></a> (SCIEX), in which the Zeno ion trap and EAD (<i>Electron-Activated Dissociation</i>) technology together make a powerful combination. It enables the creation of unique fragments to differentiate problematic amino acids and increase the sensitivity and specificity of the analysis.</p><blockquote><p><span style="font-size:22px;"><strong>We perform analyses of:</strong></span></p><ul><li>samples from solution;</li><li>samples from a gel;</li><li>samples from a complex matrix (by prior arrangement);</li><li>samples from lyophilizer;</li><li>samples previously prepared by the client.</li></ul><p>Qualitative analysis of proteins includes:</p><ul><li>To confirm the presence of a protein, the sample is subjected to digestion and the result obtained from experimental data is compared with theoretical data;</li><li>Comparison of experimental data on the masses of peptides included in the analyzed protein with the protein sequences described in the database;</li><li>Analysis and identification of the protein mixture;</li><li>Identification of post translational modifications (PTMs) (we use the tools: Skyline, Spectronaut).</li><li>Reporting of the result including, but not limited to, confirmation of the coverage of the protein sequence obtained in the test sample with the theoretical protein and the list of modified peptides, location and % of modifications.</li></ul><p><span style="font-size:22px;"><strong>Comparative analyses include:</strong></span></p><ul><li>Determining the composition of mixtures;</li><li>Comparison and profiling of sample series - protein identification and trend analysis.</li></ul><p><span style="font-size:22px;"><strong>Our offer also includes</strong>:</span></p><ul><li>Support in the planning and execution of studies (the scope is determined individually);</li><li>Assistance in the development of extraction methods from complex matrices - biological fluids, plant tissue, animal tissues (the scope is determined individually);</li><li>Statistical analysis using tools: MarkerView (PCA), GraphPad Prism, Trends, Sciex OS;</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1294"><strong>Quantitative analysis of proteins</strong></a>;</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1296"><strong>Analysis and characterization of monoclonal antibodies</strong></a>.</li></ul></blockquote><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Bioanalytic_Identification_of_the_proteins_Bioanalytic_1_97cf8c94b3.jpg" alt="Bioanalytic: Identification of the proteins." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Bioanalytic_Identification_of_the_proteins_Bioanalytic_1_97cf8c94b3.jpg 117w," sizes="100vw" width="1500" height="2000"></p>

Bioanalytic QTOF quantitative and qualitative analysis training

The purpose of the training is to introduce users to the capabilities of the LC-HR-MS/MS (QTOF) system in quantitative and qualitative analysis.

<h4><strong>BASIC INFORMATION</strong></h4><ul><li>TITLE:&nbsp;<strong> QTOF quantitative and qualitative analysis training</strong></li><li>CATEGORY: <strong>LC-MS/MS training</strong></li><li>LOCATION: <strong>Lodz, PL</strong></li><li>TRAINING TIME: <strong>3 days</strong></li><li>NUMBER OF PARTICIPANTS: <strong>up to 5 people</strong></li><li>EQUIPMENT:<strong> SCIEX</strong><span class="ts-pf-details-content"><strong> </strong></span><strong>QTOF LC-MS/MS</strong></li></ul><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Bioanalytic_Bioanalytic_QTOF_quantitative_and_qualitative_analysis_training_af1620ae8c.jpg" alt="Bioanalytic: Bioanalytic QTOF quantitative and qualitative analysis training." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Bioanalytic_Bioanalytic_QTOF_quantitative_and_qualitative_analysis_training_af1620ae8c.jpg 221w," sizes="100vw" width="1000" height="707"></p><h4><strong>TRAINING COURSE DESCRIPTION</strong></h4><p><strong>Qualitative and quantitative analysis of QTOF</strong> - the training is designed for laboratory personnel using <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?instrumentationItems=30&amp;mainauthorItems=55"><strong>high-resolution (HR) LC-MS/MS</strong></a> systems for qualitative and quantitative analysis. The following topics are covered during the training:</p><ul><li><strong>QTOF spectrometer calibration</strong></li><li><strong>Targeted and untargeted methods</strong></li><li><strong>Quantitative analysis (TOF MS, MRMhr)</strong></li><li><strong>Qualitative and quantitative analysis - DDA (IDA) vs DIA (SWATH) methodology</strong></li><li><strong>Software and its use in high-resolution data analysis</strong></li><li><strong>Reporting of results</strong></li></ul><p>The purpose of the training is to introduce users to the capabilities of the LC-HR-MS/MS (QTOF) system in quantitative and qualitative analysis. To point out the advantages of high resolution in identifying or confirming the presence of substances in a sample and also the strengths of modern QTOF systems in parallel quantitative analysis.</p><h3><strong>Training program</strong></h3><h4><span style="font-size:20px;"><strong>Day I</strong></span></h4><h5><strong>Theoretical part</strong></h5><ul><li>The principle of operation and capabilities of the QTOF system</li><li>Comparison of typical analytical procedures and their application</li><li>Targeted and quantitative analysis (TOF MS vs MRMhr)</li><li>DDA (IDA) vs DIA (SWATH) methodology.</li><li>Supporting software - Molecule Profiler, ACD Labs, MarkerView</li><li>General principles of compound fragmentation</li></ul><h4><span style="font-size:20px;"><strong>Day II</strong></span></h4><p><strong>Objective</strong> - to develop methods for quantitative analysis, confirmation or search for metabolites of a known substance, general characterization of the sample.</p><h5><strong>Practical part:</strong></h5><ul><li>Creation and optimization of the MRMhr targeted method</li><li>Creation and optimization of a non-targeted method IDA</li><li>Creation and optimization of the non-targeted method SWATH</li><li>Data acquisition (overnight)</li></ul><h4><span style="font-size:20px;"><strong>Day III</strong></span></h4><p><strong>Objective</strong> - quantitative analysis, confirmation of the presence or identification of metabolites of a known substance, general characterization of the sample (identification of compounds present in the sample), profiling.</p><h5><strong>Practical part:</strong></h5><ul><li>Quantitative analysis - TOF MS vs MRMhr</li><li>Methods for searching and analyzing data</li><li>IDA vs SWATH data</li><li>Manual search and identification</li><li>Automated search and identification - SciexOS, databases</li><li>Molecule Profiler, MarkerView</li><li>Summary and discussion</li></ul>

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