The emerging technology of mass spectrometry-based quantitative proteomics provides a powerful tool to quantitatively and
systematically assess quantitative differences in protein profiles of distinct samples and is increasingly becoming a significant component
of biomedical and clinical research. Here we provide the following workflows for protein quantification:
Gel Based Proteomics
High-throughput gel based proteomics approaches for biomarker discovery and validation.
NGS Applications
2DE-MALDI Basedadd
Two-dimensional gel electrophoresis as an excellent method for one step fractionation of complex mixture of proteins into
discrete components allowing for study of differential protein expression followed by MALDI TOF TOF protein identification in
GEL BASED PROTEOMICS approaches.
Sample Preparation for 2 DE:add
Sample Preparation for 2 DE: An important step as special care is to be taken to prepare samples for 2DE since salts, buffers
etc interfere in sample migration in the first dimension. Near to perfect sample protein quantitation will be ensured to reduce gel to gel variations.
Two Dimensional Gel Electrophoresis ( 2DE):add
Two Dimensional Gel Electrophoresis ( 2DE): Protein profiling using first, IEF on IPG strips and second SDS PAGE dimensioned on protein isoelectric point and size respectively, for 7, 11 and 24 cm format gel for superior resolution.
Differential gel electrophoresis (DIGE):add
Two Dimensional Gel Electrophoresis ( 2DE): As labeled 2DE based technique used with fluorescently labeled proteins
along with internal standard to increase 2DE sensitivity and decrease gel to gel variation during image analysis.
Image Analysis:add
2DE gels are scanned densitometric, analyzed using Image Master Platinum 7 software (GE Healthcare) for identifyingstatistical significance of differentially expressed proteins.
MALDI:add
Differentially expressed 2DE spots are excised digested with trypsin and identified by MALDI TOF TOF analysis.
1D- ESI- Q TOF LC MS MS Basedadd
Biomarker discovery and identification of proteins in immuno-precipitated samples and their interacting partners.
Sample Preparation:add
Sample Preparation for 2 DE: An important step as special care is to be taken to prepare samples for 2DE since salts, buffers etc interfere in sample migration in the first dimension. Near to perfect sample protein quantitation will be ensured to reduce gel to gel variations.
Digestion:add
The whole protein stained band is cut and digested with trypsin.
LC Separation and ESI-MS MS Analysis:add
The tryptic peptides are separated, analyzed on nano RP-UPLC connected to ESI QTOF mass spectrometry for MS and MSMS analysis.
Bioinformatics:add
The raw data is processed through the PLGS search engine for protein identification and expression. II. Gel Free Proteomics
Gel Free Proteomics
Absolute Quantificationadd
This MS workflow is used for validation of proteomic biomarkers either by
MRM method via evaluating the EIC
By adding an internal standard and estimating the absolute quantity of the unique peptides in the samples.
iTRAQ Labelingadd
This quantification MS-Workflow is a novel as well as reliable multiplexing approach (4-plex or 8-plex), most suitable for relative comparison and absolute quantification of the peptides.
chevron_rightIsobaric Labeling is done for enzymatically digested peptides at the primary amine sites (Example: 114: Control, 115,116,117: Treated/Diseased samples) which are combined together into one sample mixture.
chevron_rightThese pooled samples are purified using HPLC (SCX column) then the fractions are run on LC-MSMS (ESI -QTOF) using
Data Dependent Acquisition methodology for both Identification and Quantification.
chevron_rightThe Fraction's Raw Data generated by QTOF are analyzed using MASCOT software (MATRIX SCIENCE), which gives
normalized relative ratios for the proteins identified using ION Abundance Counts.
chevron_rightThe iTRAQ runs performed in triplicates provide robust statistics with optimized analysis.
chevron_rightBioinformatics provision: Hypothesis Testing i.e. t-test, ANOVA, Regression-Correlation value, PCA Analysis, Heat-maps,
Pie charts, coefficient variation histograms, scatter plots for spectral intensities and biological classification and functional
annotation.
Label Free Quantificationadd
A method in MS, which determines the relative amount of proteins in two or more biological samples based on their relative spectral intensity.
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Two or more biological samples with different conditions e.g. Disease vs Treated or different environmental conditions run
on LC-MS/MS (QTOF) instruments in triplicates using data independent acquisition methodology.
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Peptides are typically detected at MS1 level and distinguished from chemical noise through their characteristic isotopic
pattern i.e. different charges. The total ion current of the peptides, with confirmation from their MS2 fragments used to obtain
the spectral counts and signal intensity.
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Each sample is run separately, subsequently each peptide is mapped based on their m/z ratios and Retention Time. The
software PLGS (Waters Analysis) facilitates the identification and expression analysis, moreover normalizes and optimizes
each signal providing an accurate expression ratio of identified proteins.
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Bioinformatics provision: Hypothesis Testing i.e. t-test, ANOVA, Regression-Correlation value, PCA Analysis, Heat-maps,
Pie charts, coefficient variation histograms, scatter plots for spectral intensities and biological classification and functional
annotation.
