Biochemistry Department adds new equipment and service offerings

The characterization of large molecules requires specialized equipment. The Biochemistry Department here at Lancaster Laboratories continues to add instrumentation and service offerings to support this type of work. Thermo Scientific LTQ Orbitrap XL (LC/MS) The Orbitrap is the ultimate protein identification and biomarker discovery platform featuring unsurpassed sensitivity in MS and MS, rapid scan rate, high mass accuracy and up to 100,000 resolution power. This state-of-the-art, hybrid mass spectrometer combines a linear ion trap MS and Orbitrap mass analyzer. This system has the capability to perform comprehensive, accurate identification of proteins in complex sample mixtures. We will be using this instrument for peptide mapping, peptide sequencing, determination of post-translational modifications, proteomics, and glycan analysis. Applied Biosystems Voyager DE PRO Workstation (MALDI-TOF) Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) is an important tool for analyzing and characterizing large biomolecules of varying complexity. It’s a soft ionization technique wherein, a light absorbing matrix and a biomolecule are irradiated by a laser under vacuum. The ionized biomolecules are then introduced electrostatically to the mass spectrometer, where they are separated from the matrix ions and individually detected. The method is used for detection and characterization of proteins, peptides, oligosaccharides and oligonucleotides, with molecular masses between 400 and 350,000 daltons. It is a very sensitive method, which allows the detection of low quantities of sample with high accuracy. Protein can be identified by this technique in just a few minutes with small sample volumes. It can also provide information on microheterogeneity (e.g. glycosylation) and presence of impurities. Beckman Coulter ProteomeLab PA 800s (CE) This instrument resolves and quantifies proteins by their isoelectric point and molecular weight and generates high-resolution peptide maps and carbohydrate profiles. SDS-MW (Sodium Dodecyl Sulfate-Molecular Weight analysis) and cIEF (Capillary Isoelectric Focusing) yield critical characteristics of proteins. CE offers the advantage of high speed, great efficiency, as well as the requirement of minimum amount of sample and buffer for the analysis of protein. We hope to interface this instrument with one of our mass spectrometers in the future. Also cIEF in combination with mass spectrometry can be used to determine the identity and relative quantity of isoforms, thereby allowing remarkable advances to be made in separation of proteins and peptides. Wyatt DAWN EOS Light Scattering Instrument (MALS) This equipment is used in conjunction with various HPLC systems to perform SEC-MALS (Size Exclusion Chromatography - MultiAngle Light Scattering) analysis on protein samples. It can be used to measure and monitor protein aggregates and to characterize PEGylated and glycosylated proteins. Waters Acquity (UPLC) Ultra performance liquid chromatography (UPLC) is a new type of liquid chromatography with increased resolution, speed, and sensitivity. We added a second Waters UPLC dedicated to Peptide Mapping and HPLC method development. The other instrument has been dedicated to amino acid analysis (AAA). Peptide mapping requires shallow gradients with long run times. At optimal separation conditions, UPLC peptide maps show more resolution than other methods and increased sensitivity. UPLC can generate particularly good peak shapes for glycopeptides and find good resolution for deamidated peptides. The higher resolution and sensitivity with UPLC are particularly important when using the peptide map to detect modified peptides. Higher resolution ensures that modified peptides are resolved from the unmodified form, as well as from other peptides in the digest. Higher sensitivity means that modified peptides can be detected at lower levels.