mass spectrometry
DESCRIPTION
Mass Spectrometry . David Graham, Ph.D. [email protected] Jennifer Van Eyk , Ph.D. jvaneyk1@ jhmi.edu. Lab Goals. Familiarization with how to manipulate finished data sets and extract biological meaning Familiarization with some of the bioinformatics tools - PowerPoint PPT PresentationTRANSCRIPT
O
O
HO
HO
HOAcNH
Mass Spectrometry
David Graham, Ph.D. [email protected]
Jennifer Van Eyk, [email protected]
O
O
HO
HO
HOAcNH
Lab Goals
• Familiarization with how to manipulate finished data sets and extract biological meaning
• Familiarization with some of the bioinformatics tools• Generate a figure based upon the data
O
O
HO
HO
HOAcNH
Comparative Heart Region Proteome
• Comparative study of Rabbit heart regional proteome emphasizing on the functionally critical proteins (Calcium channels, Calcium handling protiens, Kinases, Signaling, Receptors etc.)
• Tissues isolated from 5 different tissue regions (Left ventricle, Right Ventricle, Left Atrium, Right Atrium, Septum), 3 technical replicates
O
O
HO
HO
HOAcNH
Sample Preparation and Acquisition
• Sample Preparation:– Heart regions were carefully disected rinsed in ice cold PBS and snap
frozen in liquid nitrogen– Pulvarized with a morter and pestle under liquid nitrogen– Solubilized in 8M Urea 4% Chaps– TCA (in acetone) precipitation– Multiple Acetone washes
• Digestion:– Pellet resuspended in 8M urea for 1 hours– Diluted to 2M urea– Digested with 1:100 trypsin:protein following Rapigest (Waters)
protocol without rapigest• Instrumentation:
– AB Sciex 5600 in IDA mode (data dependent discovery) choosing 40 precursors per second
– 150 uM ID external column 180 minute gradient
O
O
HO
HO
HOAcNH
Search Details
Database: Swissprot mammalsData Search: Mascot 2.3Parameters:Mass tolerance: 50 ppm, 0.1 Da ms/msModifications: Acetylation, Carbamidomethylation, Deamidation,
Carbamylation and Oxidation
Post data import into Scaffold 4.0
O
O
HO
HO
HOAcNH
Question 1: Survey your data
• Using Scaffolds built in functions:– Determine the reproducibility of your samples– Construct a venn diagram comparing replicates
O
O
HO
HO
HOAcNH
Question:2
• Data Normalization; find a common protein represented in all samples and normalize the data with it– Do statistics on data using Excel comparing heart regions (T test)– Find the proteins that are differently expressed in the samples– Identify the common and unique proteins among all three
samples. Represent it with a venn diagram
O
O
HO
HO
HOAcNH
Question:3
• Data Annotation; find the biological process, and molecular functions of the data and classify the data accordingly. Create a venn diagram for the annotation– Classify the proteins into membrane and soluble proteins– Find the potential membrane proteins and classify them based on
function. For eg. enzymatic proteins (Kinases, Dehydrogenase), structural proteins, channel proteins, receptor proteins. Create a bar graph with this data
O
O
HO
HO
HOAcNH
Question:4
• Pathway and Functional relationship; use a pathway explorer tool to generate the functional association of genes and build relationship between genes.– String (for this lab)Others:– or IPA tool for gene association– Cytoscape, Pathview
O
O
HO
HO
HOAcNH
Tools for Analysis
O
O
HO
HO
HOAcNH
Tools.
Question 2:• Excel, xlstat• Databases: Uniprot
Question3
• TMHMM Server for trans membrane predictionhttp://www.cbs.dtu.dk/services/TMHMM/• Databases: Uniprot
Question 1:• Scaffold
O
O
HO
HO
HOAcNH
Question3: Tools for data analysis: DAVIDhttp://david.abcc.ncifcrf.gov/
O
O
HO
HO
HOAcNH
Question3: Tools for data analysis: STRINGhttp://string-db.org/