journal entry #14: SEQUEST and XTandem! are proteomic search engines designed to
ID: 88177 • Letter: J
Question
journal entry #14: SEQUEST and XTandem! are proteomic search engines designed to match experimental spectra to theoretical spectra. Use the help information for each of these resources to determine the parameters each of these programs use to validate a spectral match. For example describe how XCorr and delta Cn are computed. How can XCorr or delta Cn be used to validate spectral matches? Which statistical parameters are used by XTandem! to validate spectral matches? How does XTandem! compare to SEQUEST? Journal entry #15: The MSDigest tool on the Protein Prospector website allows for the prediction of tryptic digestion sites for any protein. Choose any protein that interests you and use the MS digest tool to identify the m/z value of all of the doubly charged (2+) tryptic peptides for this protein. Include potential phosphorylation sites (S, T and Y) as a modification for this protein. Paste the output below. Journal entry #16: The role of ion trap mass spectrometry is often to identify molecular biomarkers of human disease. Many different types of biomolecules have been identified as biomarkers of of disease. A summary of such biomarkers is provided in the following review article: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4705754/ Describe 2 of the molecular biomarkers used to diagnose human disease. Which disease are these biomarkers associated with and what technologies are used for their analysis? Journal entry #17: Prosite is a database containing structural information for many proteins. Access the Prosite entry for von Willebrand factor (vWF), a protein involved in blood clotting. List some of the structural domains of vWF and describe the different functions for these structural domains. Expert Answer
Explanation / Answer
1. Xcorr or cross correlation is the measure of goodness of fit of experimental peptide fragments to theoretical spectra and Delta Correlation or delta Cn is the measure of the specificity of the fit.
Xcorr is a search dependent score. It scores the number of fragment ions and calculates the cross-correlation score for all candidate peptides queried from the database by SEQUEST searches. The higher is th score the better is the search.
Delta Cn is the normalized score difference between the current PSM(total number of identified peptide sequences for the protein) and the highest scoring PSM for that spectrum. The lower is the score the better is the search.
X! Tandem open source is software that can match tandem mass spectra with peptide sequences. The software is has a simple application programming interface(API). It simply takes an XML file of instructions on its command line and outputs the result into an XML file specified in the input.
A number of software applications e.g. Mascot, SEQUEST and MaxQuant are available to identify peptides from mass spectra.
2. Usually the MS-Digest output lists singly charged peptides. If Report Multiple Charges option is checked the output will also include multiple charged peptides. The maximum number of charges a peptide can have is based on the number of basic amino acids.
The parameter Minimum Fragment Mass specifies minimum m/z fragments in the MS-Digest report. If all fragments are to be reported the value should be set as zero.
The Maximum Fragment Mass parameter specifies the maximum m/z fragments in the MS-Digest report.
Example: The enzymatic digestion of Trypsin and endoproteinase Lys-C of glycated HAS and comparison with the digestion products of unglycated protein. Experiments were performed on doubly charged ions and resulting data was compared with the theorectical calculations.
The comparison of two spectra showed that the glycated HSA sample is a mixture of proteins with different glycation levels. As the condensation of one glucose molecule leads to increase in mass of 162 Da the mean m/z value corresponds to proteins with 14 glucose units.
3. The biomarker discovery has led to the development of clinical diagnostic tests. These tests can provide early intervention when a patient appears to be healthy(cancer and cardiovascular disease) and aid in decision making with improved clinical outcomes.
Mass spectrometers have improved technologically and can directly analyze any biological molecule susceptible to ionization. Studies on metabolomics and proteomics have helped few biomarkers to be translated into clinical tests.
For example; protein biomarker albumin can be used as nutritional marker, alanine aminotransferase can be used as marker for liver dysfunction.
One of the most promising demonstrations of mass spectrometry is in our understanding of cancer. One of the examples of small molecule metabolite biomarker is: mutation in isocitrate dehydrogenase alters its catalytic activity and result in production of oncometabolite(R)-2-hydroxygularate which is produced at very low levels in healthy cells.
4. The vWFC domain is named after von Willebrand factor(VWF) type C repeat found twice in multidomain protein. It has length of 70 amino acids with 10 well conserved cysteines. Human von Willebrand factor is a multifunctional protein involved in maintaining homeostasis.
It consists of 4 VWFD domains, 3 VWFA domains, 3 VWFB domains, 3 VWFC domains, an X domain and a C-terminal cysteine knot.
The vWF domain is found in various plasma proteins; complement factors B, C2, the integrins, collagen types and other extracellular proteins. Majority of aWF containing proteins are extracellular but the most ancient ones are intracellular involved in functions like transcription, DNA repair etc. Proteins that incorporate vWF domains take part in various biological events like cell adhesion, signal transduction etc.
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