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Biomolecule Purification, Characterization, and Analyses Biomolecule Purification, Characterization, and Analyses Characterization, Purification, Biomolecule Biomolecule Purification, Characterization, and Analyses Biomolecule Purification, Characterization, Analyses and “Quality is always about having a consistent product.” ~ Aoife Hayes, Technical Service Manager, Wexford, Ireland 239 Benchmarking, Method Development, and Troubleshooting: MassPREP Contents Peptide Standard .......................................................273 Phosphorylated Peptide Standards and Sample Preparation Kits..........................273 Innovative HPLC, UHPLC, Delta-Pak HPLC and UHPLC and UPLC Chemistry Consumables Columns ..............................................................................274 for Bioseparations ....................................................241 RapiGest SF Surfactant for Protein Digestions .....................................................275 Amino Acids ................................................................242 AccQ•Tag Ultra Derivatization Protein Separations ...............................................276 Reaction ..............................................................................242 ACQUITY UPLC SEC System Solution ..276 AccQ•Tag Amino Acid Analysis Turn Key Solution .......................................................243 Waters Insulin HMWP HPLC and UHPLC Columns .............................................278 Glycans and Glycoproteins ..............................247 XBridge Protein BEH SEC, 125Å, HILIC for Released 200Å, and 450Å Columns and N-Glycan Analysis .....................................................248 Protein Standard Test Mixtures ................... 280 HILIC for Large Molecule Benchmarking, Method Development, Glycoprotein Analysis ...........................................250 and Troubleshooting: BEH SEC Protein Standards ..................................................... 280 Benchmarking, Method Development, Protein BEH C , 300Å Columns ....................282 and Troubleshooting: Glycoprotein 4 Performance Test Standard .............................252 Benchmarking, Method Development, Released N-Glycan Sample and Troubleshooting: MassPREP Preparation with GlycoWorks ........................253 Protein Standard Mix ..............................................284 Benchmarking, Method Development, ACQUITY UPLC Glycoprotein and Troubleshooting: Glycan BEH Amide, 300Å Column ................................285 Performance Test Standards and Lifetime Testing of ACQUITY UPLC Dextran Calibration Ladders ...........................255 Glycoprotein BEH Amide, 300Å, Columns for Monosaccharide 1.7 µm Columns for Profiling IgG and Sialic Acid Analyses Subunit Glycoforms .................................................286 from Glycoproteins ...................................................256 MassPREP On-Line Desalting Devices ......................................................288 DNA, RNA, and Oligonucleotides ............... 258 IEX Cation and Anion Test Standards ....289 Columns for Large Protein-Pak Hi Res Ion-Exchange DNA/RNA Species ...................................................260 (IEX) Columns for ACQUITY UPLC Gen-Pak FAX Applications ....................................................................290 Anion-Exchange Columns ...............................260 Application of Waters UPLC Benchmarking, Method Development, Technology for Biotherapeutic and Troubleshooting: MassPREP Characterization ...........................................................291 Biomolecule Purification, Characterization, Analyses and Oligonucleotide Standard...................................261 Protein-Pak Hi Res HIC Column Oasis µElution Plates ...............................................261 and HIC Protein Standard .................................292 BioSuite HPLC Columns for Protein Peptide Separations ..............................................262 and Peptide Separations ....................................293 Peptide BEH C (130Å, 300Å), Peptide Protein-Pak High Resolution (HR) 18 Ion-Exchange Glass Columns ...................... 295 CSH C18 (130Å), and Peptide HSS T3 (100Å) Columns ............................................................262 Advanced Purification (AP) Glass Columns ............................................................. 296 Peptide CSH C18, 130Å Columns ..................264 Benchmarking, Method Development, Advanced Purification (AP) Glass and Troubleshooting: Cytochrome c Column Accessories and Spare Parts.. 296 Digestion Standard ..................................................272 Therapeutic Peptide Method Sample Preparation with ProteinWorks for Development Kit .........................................................272 Large Molecule Quantification ..................... 298 240 Biomolecule Purification, Characterization, and Analyses Innovative HPLC, UHPLC, and UPLC Chemistry Consumables for Bioseparations Advances in genomics, proteomics, metabolomics, and molecular and system biology increase our understanding of biological processes. Scientific and technological progress have revolutionized the diagnosis and treatment of disease, and it continues to do so. A leading supplier of analytical instrumentation, software, chemistry products, and services and support, Waters is uniquely positioned to provide researchers the technological means to tackle substantial analytical challenges that various biomolecules present. Keenly aware of today’s challenges, our scientists and engineers are committed to the relentless pursuit of innovative, intelligent, imaginative solutions that manifest themselves in a range of problem solving applications, from proteomics and biomarker discovery to the commercialization of advanced biopharmaceuticals. We continue to develop columns and sample-preparation consumables that support the HPLC, UHPLC, UPLC, and LC-MS analysis of peptides, proteins, oligonucleotides, amino acids, and glycoprotein associated glycans. Our comprehensive family of chemistries and consumables includes these offerings: ■■Peptide columns for nano, capillary, analytical, and preparative peptide applications ■■Protein size-exclusion, ion-exchange, hydrophobic-interaction, hydrophilic-interaction, and reversed- phase columns for analytical HPLC, UHPLC, UPLC, and laboratory-scale purification applications ■■AccQ•Tag™ Ultra chemistry, specific for Waters UPLC Amino Acid Analysis Solution as well as Pico•Tag® and AccQ•Tag chemistries for HPLC-based amino acid analyses ■■Oligonucleotide columns for synthetic oligonucleotide and DNA/RNA fragment isolations and analyses ■■ACQUITY UPLC Glycoprotein BEH Amide 300Å Columns for the analyses of intact glycoproteins, glycoprotein fragments, and glycopeptides ■■Above columns designed and quality-control tested with relevant biomolecules to help ensure column-to-column consistency ■■GlycoWorks™ RapiFluor-MS® sample preparation kits, standards, and Waters Glycan BEH Amide 130Å Columns for the analysis of released glycans ■■Analytical standards and reagents consumables and kits for MS and LC-MS applications of peptides, proteins, and other biomolecules 241 Amino Acids The constituents of proteins, amino acids are the intermediates in many metabolic pathways. Qualitative and quantitative amino acid analysis (AAA) is used to determine the concentration of proteins, identify proteins, and detect structural variants. Amino acid composition is a critical component of the nutritional value of foods and feeds. The same analytical tools are used to monitor cell culture and fermentation processes. AAA is also used as a clinical diagnostic tool for assessing inborn errors of metabolism and nutritional status. Reversed-phase chromatography provides good selectivity for separating amino acids. The most common approach to reversed- phase AAA includes pre-column derivatization. The derivatized amino acids are better retained on the reversed-phase column and are, therefore, more easily separated. Most common derivatization reagents react with amines. Some react only with primary amines, but the most useful ones also react with secondary amines so that proline and hydroxyproline are also measured. In addition to improving chromatography, derivatization can render amino acids readily detectable by UV absorbance or fluorescence. Waters offers an UPLC as well as HPLC-based method for AAA that employ pre-column derivatization and reversed-phase chromatography for accurate identification and quantitation of free or amino acids hydrolyzed from protein digests: AccQ•Tag (HPLC-based) and AccQ•Tag Ultra (UPLC-based). Hundreds of published papers report the successful application of our AccQ- Fluor pre-column chemistry for amino acid derivatization. The stable derivatized AAs are then resolved on our AccQ•Tag or AccQ•Tag Ultra C18 Column with on-line detection of the resolved amino acids, using UV absorbance or fluorescence detection. ACCQ•TAG ULTRA DERIVATIZATION REACTION The following steps describe the AccQ•Tag Ultra derivatization process: ■■ Starting material is AccQ•Tag Ultra Reagent Powder ¡■ 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) ¡■ US Patent #5,296,599 and European Patent #EP 0 533 200 B1 ■■ AQC reacts rapidly with both primary and secondary amines ■■ Excess reagent reacts more slowly with water, forming 6-aminoquinoline (AMQ) ■■ AMQ reacts slowly with excess AQC reagent, forming a bis urea ■■ Derivatized amino acids are separated chromatographically from the by-products ■■Requires no vacuum drying, sample prep, or extraction Chemistry of the AccQ•Tag Derivatization Reaction O O O OH 1° Amino Acid: OH H t <<1s H N O NH2 1/2 N NH
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