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Protein Mass Fingerprinting Protein Profiling Protein assay
BIO105 / AC1 ____________________________ BY: VIOLETA, RAYMUNDO, LEE, GOYON, DOLINA, MARQUEZ

Protein Mass Fingerprinting
• Protein Mass Fingerprinting (PMS) is a technique for protein and peptide identification

• One method of improving the specificity of a peptide mass fingerprint was first proposed by Peter James

Protein Mass Fingerprinting
• Advantage : only the masses of the peptides have to be known • Disadvantage : - the protein sequence has to be present in the database of interest - most PMF algorithms assume the peptides come from a single protein

Protein Mass Fingerprinting
digest MS Spectrum processing
Protein (gel band or spot)
peptides Mass spectrum

1554.25 2055.39 1942.44 1755.67 987.55 855.34 677.68

Peak list

Search

Protein X theoretical digest

Protein Y theoretical digest

Protein Z theoretical digest

Report
HIT Protein X Protein Y Protein Z SCORE 1000 50 5

Protein Mass Fingerprinting
• Mass spectrum is a plot of m/z versus intensity • A mass spectrum of the peptide mixture resulting from the digestion of a protein by an enzyme provides a fingerprint of great specificity. • This method of identification is much more reliable than using fingerprints based on PAGE migration patterns or HPLC retention times.

Protein Mass Fingerprinting
• SDS-PAGE (sodium dodecyl sulphatepolyacrylamide gel electrophoresis) is commonly used in the lab for the separation of proteins based on their molecular weight.



HPLC retention times depends on the separation mode that you are using. More hydrophilic (polar) compounds will stay on the column longer and have a larger retention time.

Protein Mass Fingerprinting
Simple Mass Spectrum
Intensity
771.9 1165.2 361.4 320.3 607.7 992.1

681.7

200

400

600

800

1000

1200

1400

1600

Mass (Da)

320.3 361.4 607.7 681.7 771.9 992.1 1165.2
List of masses

Database search using computer algorithm

e.g. MASCOT

EAT EGR ISPYK EMETR EMANYK PLEASEMAK EATSTHEYAR
Sequence matches

PLEASEMAKEMANYKRISPYKREMETREATSTHEYAREGREAT

Protein Mass Fingerprinting
• MALDI TOF Mass Spectrum
Figure 1.

Protein Mass Fingerprinting
• Mass Spectra are acquired with..
MALDI TOF MS (Voyager DE PRO, ABI) MALDI TOF/TOF MS (4700 Proteomics Analyzer, ABI)

MALDI – Matrix Assisted Laser Desorption Ionization
TOF – Time Of Flight MS – Mass Spectrometry

Protein Mass Fingerprinting
Peak List • Spectrum viewer • Compiled from the mass spectra
– Mass list – Mass list and intensity

• Peak list is submitted for Database searching

Protein Profiling
• Clinical Proteomics is the application of proteomic technologies to molecular profiling of tissues or body fluids in all aspects of clinical research • Clinical Proteomics workflow is very complex, starting from upstream specimen’s acquisition, storage, sample preparation.

Protein Profiling
• Protein Synthesis is the most useful technique that allows formation of reference databases for cells and tissues and performance of comparative proteomics • still used today in many applications including characterization of new organisms and viruses.

Protein Profiling
2D DIGE Protein Expression Profiling Procedure
1. Sample preparation 2. Sample labeling with CyDye DIGE fluors 3. 2D gel electrophoresis 4. Image analysis 5. Quantitative analysis 6. Automated spot picking 7. Cluster Analysis 8. Validation

Protein Profiling
• Protocol protein extraction from tissues is followed by 2D gel electrophoresis (2DE) with subsequent in-gel digestion and identification of soluble proteins by two individual mass spectrometric techniques, tandem matrixassisted laser desorption/ionization mass spectrometry (MALDI-MS) and nano-liquid chromatography (nano-LC)-MS/MS. The proposed combined use of these two MS approaches leads to a very high identification rate of well-separated protein spots from a gel.

Protein Profiling
• In the first step 2DE separates highabundance proteins (those visualized by nonsensitive Coomassie blue staining) that are subsequently picked, digested and aliquoted for MS applications. Protein samples not identified by MALDI-MS or MS/MS (77% of all spots) are finally unambiguously identified by nano-LC-MS/MS (total identification rate 94%). This protocol can be completed in 6 weeks.

Protein Profiling
• Identifying the proteins expressed in a particular tissue, under a specified set of conditions and at a particular time, usually compared to expression in reference samples • Is useful in drug discovery and diagnosis as well as in understanding response mechanisms at the protein level

Protein Profiling
• A method, based on machine learning, for isolating the sets of proteins, before identifying them by name, which classify accurately the treatment classes in a study • If proteins associated with known classes of interest it can be used to identify unknown classes then the proteins are definitive for diagnosis.

Protein Profiling
• Multiple two-dimensional electrophoresis (2DE) gel patterns are included in each treatment class • The proteins in each class, including controls, are converted to digital data and serve as input to artificial neural network (ANN) models. Multiple two-dimensional electrophoresis (2DE) gel patterns are included in each treatment class. A training subset of digitized individual, not composite, gel images is used to construct an ANN model which is then applied to a test set of images.

Protein Assay
•An assay for a protein is simply a method of determining in a quantitative fashion the amount of a particular activity present. •Assay is an investigative (analytic) procedure in laboratory medicine, pharmacology, environmental biology, and molecular biology for qualitatively assessing or quantitatively measuring the presence or amount or the functional activity of a target entity (the analyte), which can be a drug or biochemical substance or a cell in an organism or organic sample.The measured entity is generally called the analyte, or the measurand or the target of the assay

Protein Assay
Types of protein assay: 1. Absorbance Assays - Absorbance at 280 nm - Absorbance at 205 nm - Extinction Coefficient 2. Colorimetric Assays - Modified Lowry - Biuret - Bradford Assay - Bicinchoninic Acid (Smith) - Amino Black Method - Colloidal Gold

Protein Assay
• Absorbance Assay Principle Proteins in solution absorb ultraviolet light with absorbance maxima at 280 and 200 nm. Amino acids with aromatic rings are the primary reason for the absorbance peak at 280 nm. Peptide bonds are primarily responsible for the peak at 200 nm. Secondary, tertiary, and quaternary structure all affect absorbance, therefore factors such as pH, ionic strength, etc. can alter the absorbance spectrum.

Protein Assay
Equipment In addition to standard liquid handling supplies a spectrophotometer with UV lamp and quartz cuvette are required.

•Colorimetric Assay Principle Colorimetric assays use reagents that undergo a measurable color change in the presence of the analyte. They are widely used in biochemistry to test for the presence of enzymes.

Protein Assay
Equipment Spectrophotometer - a photometer that can measure intensity as a function of the light source wavelength. Important features of spectrophotometers are spectral bandwidth and linear range of absorption or reflectance measurement.

Protein Assay
Important criteria for choosing an assay include: • Compatibility with the sample type and components • Assay range and required sample volume • Protein-to-protein uniformity • Speed and convenience for the number of samples to be tested • Availability of spectrophotometer or plate reader necessary to measure the color produced (absorbance) by the assay

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