scholarly journals Advantageous Uses of Mass Spectrometry for the Quantification of Proteins

2013 ◽  
Vol 2013 ◽  
pp. 1-6 ◽  
Author(s):  
John E. Hale
Keyword(s):  
Mass Spectrometry ◽  
Great Difficulty ◽  
Immunological Methods ◽  
Regulatory Agencies ◽  
Protein Species ◽  
Sample Handling ◽  
Wide Acceptance ◽  
Immunological Assays ◽  
Protein Assays ◽  
Very High

Quantitative protein measurements by mass spectrometry have gained wide acceptance in research settings. However, clinical uptake of mass spectrometric protein assays has not followed suit. In part, this is due to the long-standing acceptance by regulatory agencies of immunological assays such as ELISA assays. In most cases, ELISAs provide highly accurate, sensitive, relatively inexpensive, and simple assays for many analytes. The barrier to acceptance of mass spectrometry in these situations will remain high. However, mass spectrometry provides solutions to certain protein measurements that are difficult, if not impossible, to accomplish by immunological methods. Cases where mass spectrometry can provide solutions to difficult assay development include distinguishing between very closely related protein species and monitoring biological and analytical variability due to sample handling and very high multiplexing capacity. This paper will highlight several examples where mass spectrometry has made certain protein measurements possible where immunological techniques have had a great difficulty.

scholarly journals Antigens of worms and eggs showed a differentiated detection of specific IgG according to the time of Schistosoma mansoni infection in mice

2012 ◽  
Vol 45 (4) ◽  
pp. 505-509 ◽  
Author(s):  
Rafaella Fortini Queiroz Grenfell ◽  
Watson Hermann Martins ◽  
Vanessa Silva-Moraes ◽  
Suedali Villas-Boas Barata ◽  
Elizandra Giani Ribeiro ◽  
...  
Keyword(s):  
Schistosoma Mansoni ◽  
Experimental Analysis ◽  
Immunological Methods ◽  
Perfusion Technique ◽  
Individual Number ◽  
Specific Igg ◽  
Immunological Assays ◽  
The Individual ◽  
Positive Groups ◽  
Sera Samples

INTRODUCTION: The correlation between the immunological assay and the antibody titer can offer a tool for the experimental analysis of different phases of the disease. METHODS: Two simple immunological assays for Schistosoma mansoni in mice sera samples based on specific IgG detection for worms soluble antigens and eggs soluble antigens were standardized and evaluated in our laboratory. Fifty mice were used in negative and positive groups and the results obtained by enzyme-linked immunosorbent assays (ELISA) assays were compared with the number of worms counted and the IgG titers at different times of infection. RESULTS: Data showed that ELISA using adult worm antigens (ELISA-SWAP) presented a satisfactory correlation between the absorbance value of IgG titers and the individual number of worms counted after perfusion technique (R²=0.62). In addition, ELISA-SWAP differentially detected positive samples with 30 and 60 days post infection (p=0.011 and 0.003, respectively), whereas ELISA using egg antigens (ELISA-SEA) detected samples after 140 days (p=0.03). CONCLUSIONS: These data show that the use of different antigens in immunological methods can be used as potential tools for the analysis of the chronological evolution of S. mansoni infection in murine schistosomiasis. Correlations with human schistosomiasis are discussed.

scholarly journals Mass spectrometry analysis reveals differences in the host cell protein species found in pseudotyped lentiviral vectors

Biologicals ◽  
2018 ◽  
Vol 52 ◽  
pp. 59-66 ◽  
Author(s):  
Sabine Johnson ◽  
Jun X. Wheeler ◽  
Robin Thorpe ◽  
Mary Collins ◽  
Yasuhiro Takeuchi ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Host Cell ◽  
Lentiviral Vectors ◽  
Mass Spectrometry Analysis ◽  
Cell Protein ◽  
Protein Species ◽  
Host Cell Protein ◽  
Spectrometry Analysis

scholarly journals A streamlined pipeline for multiplexed quantitative site-specific N-glycoproteomics

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Pan Fang ◽  
Yanlong Ji ◽  
Ivan Silbern ◽  
Carmen Doebele ◽  
Momchil Ninov ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Resolution ◽  
Sample Preparation ◽  
Large Scale ◽  
Handling Time ◽  
Database Search ◽  
High Confidence ◽  
Site Specific ◽  
Sample Handling ◽  
Data Processing Tool

Abstract Regulation of protein N-glycosylation is essential in human cells. However, large-scale, accurate, and site-specific quantification of glycosylation is still technically challenging. We here introduce SugarQuant, an integrated mass spectrometry-based pipeline comprising protein aggregation capture (PAC)-based sample preparation, multi-notch MS3 acquisition (Glyco-SPS-MS3) and a data-processing tool (GlycoBinder) that enables confident identification and quantification of intact glycopeptides in complex biological samples. PAC significantly reduces sample-handling time without compromising sensitivity. Glyco-SPS-MS3 combines high-resolution MS2 and MS3 scans, resulting in enhanced reporter signals of isobaric mass tags, improved detection of N-glycopeptide fragments, and lowered interference in multiplexed quantification. GlycoBinder enables streamlined processing of Glyco-SPS-MS3 data, followed by a two-step database search, which increases the identification rates of glycopeptides by 22% compared with conventional strategies. We apply SugarQuant to identify and quantify more than 5,000 unique glycoforms in Burkitt’s lymphoma cells, and determine site-specific glycosylation changes that occurred upon inhibition of fucosylation at high confidence.

Development of open-access mock 510(k) regulatory documents on multiplex proteomic technologies.

2012 ◽  
Vol 30 (30_suppl) ◽  
pp. 99-99
Author(s):  
Emily Boja ◽  
Tara Hiltke ◽  
Chris Kinsinger ◽  
Mehdi Mesri ◽  
Robert Rivers ◽  
...  
Keyword(s):  
Open Access ◽  
Development Process ◽  
Clinical Chemistry ◽  
Regulatory Approval ◽  
Regulatory Agencies ◽  
Community Resources ◽  
In Vitro Diagnostics ◽  
Regulatory Documents ◽  
Protein Assays

99 Background: Proteome-based biomarker science is progressing slowly for a variety of factors, including a lack of dialogue early in the discovery/development process with regulatory agencies such as the Food and Drug Administration (FDA). Although high-throughput multiplex proteomic-based technologies have the potential to accurately measure proteins of clinical significance in complex human matrices, regulatory approval for these analytical technologies have not been explored. Methods: To address this gap, investigators from the National Cancer Institute’s Clinical Proteomic Tumor Analysis Consortium (CPTAC) held a forward-thinking workshop, involving clinical laboratories, instrument manufacturers, researchers, and FDA representatives in order to streamline the regulatory approval of protein-based multiplex platforms. Results: Outcomes included a peer-reviewed workshop report published in special proteomics issue of Clinical Chemistry, and first-of-its-kind open access mock 510(k) regulatory documents (based on two most commonly applied quantitative proteomic technologies: multiplex mass spectrometry-based and multiplex affinity-based platforms) as public community resources (also published in Clinical Chemistry). Conclusions: These open-access documents provide a glimpse of the FDA’s perspective of novel approaches to in vitro diagnostics of multiplex protein assays and the process of premarket review. Therefore, these resources should enable others to focus on aspects of the process carefully monitored by the FDA, observe a format that is acceptable to the agency, and understand the iterative nature of review.

scholarly journals Combiningin situproteolysis and mass spectrometry to crystallizeEscherichia coliPgaB

2012 ◽  
Vol 68 (7) ◽  
pp. 842-845 ◽  
Author(s):  
Dustin J. Little ◽  
John C. Whitney ◽  
Howard Robinson ◽  
Patrick Yip ◽  
Mark Nitz ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Escherichia Coli ◽  
Crystal Quality ◽  
Protein Species ◽  
Space Group ◽  
Protein Target ◽  
Sds Page ◽  
Initial Crystal

The periplasmic poly-β-1,6-N-acetyl-D-glucosamine (PNAG) de-N-acetylase PgaB fromEscherichia coliwas overexpressed and purified, but was recalcitrant to crystallization. Use of thein situproteolysis technique produced crystals of PgaB, but these crystals could not be optimized for diffraction studies. By analyzing the initial crystal hits using SDS–PAGE and mass spectrometry, the boundaries of the protein species that crystallized were determined. The re-engineered protein target crystallized reproducibly without the addition of protease and with significantly increased crystal quality. Crystals of the selenomethionine-incorporated protein exhibited the symmetry of space groupP212121and diffracted to 2.1 Å resolution.

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