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Mass Spectrometry
David Graham, Ph.D.
dgraham@jhmi.edu
Jennifer Van Eyk, Ph.D.
jvaneyk1@jhmi.edu
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Lab Goals
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• 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
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Comparative Heart Region Proteome
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Comparative study of
Rabbit heart regional
proteome
emphasizing on the
functionally critical
proteins (Calcium
channels, Calcium
handling protiens,
Kinases, Signaling,
Receptors etc.)
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Tissues isolated from 5
different tissue regions
(Left ventricle, Right
Ventricle, Left Atrium,
Right Atrium, Septum),
3 technical replicates
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Sample Preparation and Acquisition
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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
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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
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Instrumentation:
– AB Sciex 5600 in IDA mode (data dependent discovery) choosing 40
precursors per second
– 150 uM ID external column 180 minute gradient
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Search Details
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Database: Swissprot mammals
Data Search: Mascot 2.3
Parameters:
Mass tolerance: 50 ppm, 0.1 Da ms/ms
Modifications: Acetylation, Carbamidomethylation, Deamidation,
Carbamylation and Oxidation
Post data import into Scaffold 4.0
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Question 1: Survey your data
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• Using Scaffolds built in functions:
– Determine the reproducibility of your samples
– Construct a venn diagram comparing replicates
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Question:2
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• 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
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Question:3
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• 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
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Question:4
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• 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
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Tools for Analysis
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Tools.
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Question 1:
• Scaffold
Question 2:
• Excel, xlstat
• Databases: Uniprot
Question3
• TMHMM Server for trans membrane prediction
http://www.cbs.dtu.dk/services/TMHMM/
• Databases: Uniprot
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Question3: Tools for data analysis: DAVID
http://david.abcc.ncifcrf.gov/
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Question3: Tools for data analysis: STRING
http://string-db.org/