Proteins are a versatile group of macromolecules; peptides are smaller in size with a limited number of building stones. Both consist out of a chain of amino acids, their structure is determined not only by the amino acids, but also by interactions between them, forming a secondary and tertiary structure which is important for the activity of the molecule and its chemical and physical characteristic.

Proteins are present in all cells, functioning e.g. as enzymes, as transmitters or as biomarkers. Understanding about conditions and mechanism of their expression or absence is crucial for further development of biopharmaceuticals for a targeted therapy of diseases.

 
 
 
 

Applications for Peptides and Proteins Analysis 

 

Quality Control Analysis of Synthetic Peptides Using the MALDI-8020 Benchtop Linear MALDI-TOF Mass Spectrometer and QC Reporter Software

Within the manufacturing process of bio-therapeutics, quality control (QC) plays a fundamental role in guaranteeing the supply of a high quality product. Any changes in the product formulation or degradation can affect the therapeutic role, leading to a potential loss of activity or development of toxicity. Amongst the several analytical techniques that can be used to determine the quality of a synthetic bio-product, MALDI-TOF mass spectrometry is widely employed due to its rapid and simple operation, low running costs, sensitivity and ability to provide information on the molecular weight as well as the sequence and structure of a compound, its impurities/adducts and modification products.

 
 
 
 
 
 

Protein Analysis Platform: Combining the powerful capabilities of MALDI-TOF MS (MALDI-8020) and Edman Sequencing (PPSQ-50A Gradient System) for accurate N-terminal sequence of peptides

Mass spectrometry has become an indispensable tool for researchers looking to sequence peptides. Although effective in many cases, sequencing by In Source Decay (ISD) faces a few challenges its ability to provide reliable sequence information including isobaric amino acids, database dependency and low molecular weight interferences. Traditional Edman sequencing avoids mass dependency and the use of databases by analyzing each amino acid from the N-terminus one at a time in sequence. Unfortunately, Edman has its own limitations in providing high sequence coverage. This technical note investigates the benefits of combining the intact mass and sequencing information from the MALDI-8020 (ISD) with the N-terminal sequence obtained from the PPSQ-50A gradient system (Edman). Using the combined information allows investigators the ability to obtain a more complete picture of their proteins and peptides of interest.

 
 
 
 
 
  
 

 

Primary Structural Analysis of Proteins / Peptides – Application of Protein Sequencer –

Understanding of pathological states and life phenomena has accelerated dramatically with recent widespread advances in the field of proteomics, which encompasses the exhaustive comparison and identification of expressed proteins in normal and disease-state tissues and cells. The scope of this investigation includes pathology, differences in type, genetic variation and cell abnormalities across a range of scientific disciplines. For example, investigation of the causes of disease and treatment methods, and the search for specific biomarkers associated with those diseases in the field of proteomics has grown rapidly. The word "proteomics" was coined in the early 1990s to indicate the entire set of proteins expressed in a specific cell type or organism associated with that genome. In general, there is very little in the way of change in the genome in a given type of organism, but the change in the proteome between organs and cells can be vast. In order for an organism to make its genetic information actually function, the process of gene expression is required. 

 
 
 
 
 
 

 

Protein Sequence Analysis by In-Source Decay Using a Benchtop MALDI-TOF Mass Spectrometer

MALDI-TOF mass spectrometers are frequently used for molecular weight measurement and identification of proteins. To identify proteins using a mass spectrometer, generally tryptic digestion is required for preparation; however, if the protein is isolated and purified, by detecting the ions produced by fragmentation within an ion source (ISD, In-Source Decay), sequence analysis of intact proteins can be performed with no need to digest the protein with trypsin.

 
 
 
 
 
 

 

Detection of High-mass Proteins Using a Benchtop MALDI-TOF Mass Spectrometer

The applicability of MALDI-TOF mass spectrometry to perform protein detection is well recognized in the life science field. In this field, SDS polyacrylamide gel electrophoresis and size exclusion chromatography have been historically used, however, they have drawbacks such as being time-consuming or lacking accuracy in molecular weight determination. Due to its ability to provide more accurate molecular weight information, MALDI-TOF mass spectrometry has become the primary tool for the analysis of protein primary structures.

 
 
 
 
 
 

 

Direct Quantitation of Intact Proteins By Multiple Ion Chromatogram Method on Q-TOF Mass Spectrometer

With the growth of global protein therapeutic market, it is particularly important to accurately and precisely monitor the protein levels through out the process of production, characterization, and clinical uses. Over the last 20 years, mass spectrometry has become a powerful analytical technique for protein study. In quantifying intact proteins, selected ion monitoring (SIM) and multiple reaction monitoring (MRM) have been reported by using the triple-quadrupole (QQQ). Quadrupole time-of-flight(Q-TOF) is more sensitive and specific than QQQ, and it is commonly used in profiling analysis. However, due to a lower dynamic range, there is lack of quantitative studies for intact proteins by using Q-TOF.

 
 
 
 
 
 

 

Protein Identification from Two-dimensional Gel Electrophoresis Based on Peptide Mass Fingerprinting (PMF) Using a Benchtop MALDI-TOF Mass Spectrometer

At present, shotgun proteomics techniques using liquid chromatography mass spectrometry are utilized mainly as high-throughput methods for identifying many different proteins in cellular cytoplasm. However, these techniques are not necessarily effective for identifying all proteins. In particular, when handling proteins separated by means of two-dimensional electrophoresis etc., the protein spots detected on the electrophoresis gel must be linked to the results of protein identification. For such analyses, there may be many cases where using MALDI-TOF mass spectrometry is more efficient than using liquid chromatography mass spectrometry after enzyme treating the protein spots separated from the gel. This article introduces an example of protein identification using two-dimensional electrophoresis and a benchtop MALDI-TOF mass spectrometer.

 
 
 
 
 
 

 

Extraction of N-terminally Blocked Proteins from Electrophoresis Gels and Sequence Analysis Using a Benchtop MALDI-TOF Mass Spectrometer

Proteins may now be easily identified using trypsin digestion pretreatment and a mass spectrometer. This type of workflow is well-established and is accessible even to non-MS experts. In such workflows, protein identification is achieved through simple database searching with a search engine, such as Mascot (Matrix Science). However, in the case of terminal sequence analysis of proteins that have undergone processing or mass spectrometric identification of proteins from minor biological species not listed in databases, more expensive high-end instrumentation and more complex workflows, requiring skilled operators, are often employed. Furthermore, protein sequencers are generally used as a method for protein terminal sequence analysis; but in the case of N-terminally blocked proteins, de-blocking is necessary as a pretreatment to perform Analysis.

 
 
 
 
 
 

 

MALDI-MS Protein Profiling of Chemoresistance in Extracellular Vesicles of Cancer Cells

Faster detection of chemoresistance will improve cancer therapy and therefore improve cancer survival rates. Cancer cells communicate with the whole organism via extracellular vesicles (EVs), which circulate the body and propagate molecular information in support of the malignant phenotype. EVs were harvested from cell culture supernatant by ultracentrifugation to serve as a model for circulating cancer cell-derived biomarker carriers from body fluids (i.e., liquid biopsy). In this work, differential expression of proteins in EVs, measured by MALDI-TOF-MS, as the result of an increasing chemoresistance of their parent cells was observed.

 
 
 
 
 
 

 

Peptide Mapping of Antibody Drugs by Nexera-i

Peptide mapping by HPLC is one of the important quality assurance tests used for verifying the primary structure of antibody drugs. Typically, following enzymatic digestion of the antibodies, separation is conducted using a traditional reversed phase column. Due to the large number of peaks that require separation, the use of small-particle columns and core shell columns for peptide analysis has spread in recent years. In order to compare elution profiles for identity and mutation confirmation, a highly repeatable system is required.

 
 
 
 
 
 

 

Evaluation of Membrane Protein Properties by Fluorescence-Detection Size-Exclusion Chromatography (FSEC) Using an HPLC System

Cells, the smallest unit comprising the human body, are covered by a membrane consisting of lipids. Various membrane proteins are located in the cell membrane, receiving information from outside the cell and transporting substances inside and outside the cell. These membrane proteins have garnered attention not only because they are associated with important biological phenomena, but also as target molecules for drugs.

 
 
 
 
 

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