Abbreviations:Ab (antibody), H-CDR3 (heavy-chain complementarity determining region 3), LC (liquid chromatography), LC-MS/MS (liquid chromatography tandem mass spectrometry), MS (mass spectrometry)
- •Recognize the newest techniques in biomedical research.
- •Describe how these techniques can be utilized and their limitations.
- •Describe the potential impact of these techniques.
- •Enables direct analysis of protein amino acid sequences, allowing for identification of unknown proteins (e.g., new autoantigens in disease)
- •Enables analysis of changes in global protein expression, for example, in epidermis or other organs under different experimental conditions
- •Limits in the detection of proteins in very complex samples, requiring reduction in complexity of samples of interest (e.g., by affinity purification)
- •Nondetection of a protein of interest in complex samples does not exclude presence of the protein, and detection of a peptide characteristic for one protein may not be specific for this protein because peptides can be shared between proteins (i.e., protein interference problem
- •Experienced bioinformaticians are needed to interpret the complex MS results
Isolation and Fractionation of the Protein Sample of Interest
Bioinformatic Analysis and Interpretation of MS Data
Examples of Applications of MS in Immunology and Investigative Dermatology
Conflict of Interest
Multiple Choice Questions
- 1.In analysis and interpretation of tandem mass spectrometry (MS/MS) data, which of the following statements is not correct?
- A.Theoretical MS/MS spectra are generated from in silico analysis of predicted digestion products of known proteins.
- B.To interpret MS spectra of human antibodies (and their clonalities), a custom database or de novo interpretation is required.
- C.Precursor ion spectra are correlated with theoretical MS/MS spectra generated from protein databases.
- D.Public protein databases serve as input to guide in silico analysis of proteins into predicted digestion products.
- 2.In the fields of immunology and investigative dermatology, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been successfully used to
- A.identify an unknown protein from immunoprecipitation.
- B.characterize plasma membrane proteins within the human epidermis.
- C.characterize the circulating antibody response in an autoimmune disease and/or after immunization.
- D.All of the above
- 3.Which of the following statements about potential limitations of LC-MS/MS is not correct?
- A.Ambiguity in protein inference can be introduced by the use of proteolytic enzymes and by redundancy phenomena in the databases used for comparison.
- B.Unknown proteins can be easily identified without the use of protein databases.
- C.Different structural labilities of peptide bonds can make interpretation of MS/MS spectra difficult.
- D.De novo interpretation of fragment ion spectra is time consuming and error prone.
- 4.What are the main underlying principles that allow for separation of peptides by reverse-phase liquid chromatography (LC) and mass spectrometry (MS), respectively?
- A.Peptide hydrophobicity, only the charge of the peptide
- B.Peptide’s charge and mass in both LC and MS
- C.Only the mass in LC, the mass and the charge in MS
- D.Peptide hydrophobicity, the mass and the charge of the peptide
- 5.Which of the following statements is correct?
- A.The proteolytic enzyme trypsin cuts proteins after amino acids arginine and lysine.
- B.The heavy-chain complementarity determining region 3 (H-CDR3) is a unique identifier of an antibody and can be detected by LC-MS/MS experimentation.
- C.Collision-induced dissociation describes fragmentation of precursor ions in a collision cell and does not always result in all potential fragment ions (e.g., b- and y-ions) of a given peptide.
- D.All of the above
- Quiz and brief explanation of correct answers
- Teaching Slides
- A proteomic characterization of the plasma membrane of human epidermis by high-throughput mass spectrometry.J Invest Dermatol. 2004; 123: 691-699
- High-throughput protein analysis using mass spectrometry-based methods.School of Biotechnology, KTH Royal Institute of Technology, Stockholm2014
- Proteomic analysis of pemphigus autoantibodies indicates a larger, more diverse, and more dynamic repertoire than determined by B cell genetics.Cell Rep. 2017; 18: 237-247
- Mass spectrometry and protein analysis.Science. 2006; 312: 212-217
- Proteomic analysis of filaggrin deficiency identifies molecular signatures characteristic of atopic eczema.J Allergy Clin Immunol. 2017; 140: 1299-1309
- Laboratory diagnosis of pemphigus: direct immunofluorescence remains the gold standard.Br J Dermatiol. 2016; 175: 185-186
- Pemphigus. Nature Rev Dis Primers. 2017; 3: 17026
- Identification and characterization of the constituent human serum antibodies elicited by vaccination.Proc Natl Acad Sci USA. 2014; 111: 2259-2264
- Molecular-level analysis of the serum antibody repertoire in young adults before and after seasonal influenza vaccination.Nat Med. 2016; 22: 1456-1464
- UniProt Knowledgebase: a hub of integrated protein data.Database (Oxford). 2011; 2011 (bar009)
- Autoantibodies against glutamate receptor delta2 after allogenic stem cell transplantation.Neurol Neuroimmunol Neuroinflamm. 2016; 3: e255
- The protease inhibitor alpha-2-macroglobulin-like-1 is the p170 antigen recognized by paraneoplastic pemphigus autoantibodies in human.PLoS One. 2010; 5: e12250
- Molecular deconvolution of the monoclonal antibodies that comprise the polyclonal serum response.Proc Natl Acad Sci USA. 2013; 110: 2993-2998
- Serology in the 21st century: the molecular-level analysis of the serum antibody repertoire.Curr Opin Immunol. 2015; 35: 89-97
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