Mcp Lect 18: Mass Spectroscopy And Medical Proteomics

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Biomarker

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Any biological molecule or biochemical feature can be considered a biomarker if:
1. it can be readily measured
2. it is differentiating in some biological system
3. direct measurements of the desired biological state are impossible or too expensive.

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Systems biology, in medicine, uses biomarkers to ...

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understand the mechanisms of disease, drug resistance, progression and prognosis.

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Systems/Personalized medicine uses biomarkers to...

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guide clinical decisions for a specific patient.

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False negative v. false positive v. true positive v. true negative biomarker predictions?

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False negative biomarker predictions fail to detect a disease in people with the disease, while false positive biomarker predictions indicate the presence of a disease in people without it. true positives and true negatives are defined analogously and represent correct biomarker predictions.

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Sensitivity

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Tests with high sensitivity have few false negatives

True positive / (True positive + False Negative)

A single biomarker measurement can sometimes be made sensitive OR specific by the selection of an appropriate threshold for prediction.

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Specificity

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Tests with high specificity have few false positives

True negative / (True negative +False positive)

A single biomarker measurement can sometimes be made sensitive OR specific by the selection of an appropriate threshold for prediction.

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What characteristics of biomarkers are important in research studies? In the clinical context?

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Research: sensitivity and specificity
Clinical: positive predictive value and negative predictive value

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Positive predictive value

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the proportion of samples that test positive that have the disease

TP/(TP+FP)

particularly important statistic when considering tests for a rare disease applied to a very large population

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Negative predictive value

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the proportion of samples that test negative that do not have the disease

TN/(TN+FN)

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False positives

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= (1-specificity)(1-prevalence)(population of N)

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True positives

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(specificity)(prevalence)(population of n)

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Benefits to restricting tests to at risk populations?

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Reduces N (the population) and increases prevalence, helps with low PPV

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The use of proteins as biomarkers has a number of advantages over genomic biomarkers. In particular ...

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1. Proteins are dynamic
-->Sub-cellular location, organ, abundance, binding, assembly, cleavage, modification
2. Multiple proteins encoded in each gene
-->Specific protein product depends on the context
3. Cancer (disease) can perturb protein production and processing – observable in blood, urine, and other easily accessible bodily fluids.
4. Mass spectrometry provides unbiased, automated, data-rich measurements of proteins in clinical samples.

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What does 2-Dimensional Gels (SDS-PAGE) tell you?

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Global view of protein abundance

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What does mass spectrometry (of a protein) tell you ?

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Protein identification by Peptide Mass Fingerprinting

Peptide mass fingerprinting = the mass spectrum obtained from the peptide ions produced by the enzymatic digestion of a single protein

1. 2D gel (SDS-PAGE)
2. Cut out spot of protein of interest
3. Cut up protein into peptides --> often uses trypsin, which cuts are arginine and lysine. Peptides of 10-155 aa in length are ideal for ionization
4. Use protein sequence database to interpret peptide mass fingerprints
-->go to protein database and look at protein you think it is, and predict how that protein would have been cut up by the enzyme you used
-->see if those fragments are present on the mass fingerprint spectrum

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What does tandem mass spec tell you?

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Direct peptide sequence characterization

*dont need to cut out gel spots --> analyze whole cell lysate. Enzymatically digest (trypsin) and fractionate that sample (first separated on basis of hydrophobicity then MS of intact peptide ions is obtained) and get pool of peptides to go after. Instrument can acquire spectrum and then you can pick out particular peak as interesting (this gives you mass of intact peptide) throw out all the other ones and measure individual peaks of interest.
For the individual peak of interest (typically the most intense peptide ions), all copies of that peptide through a collision chamber full of gas. This 2nd mass spec observes the masses of PIECES of peptide. The protein fragmentation can tell you what sequence of peptide is.

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b type ions

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Fragment generated in tandem mass spec; has charge on the amino terminal side

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m/z ?

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mass to charge ratio

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y type ions

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Fragment generated in tandem mass spec; has charge on the carboxyl terminal side