Sandor LifeSciences
Biosciences Research Services

Quantitative Proteomics

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.

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.

An important step as special care is to be taken to prepare samples for 2DE since salts, buffers etc interfere in sample migration in first dimension. Near to perfect sample protein quantitation will be ensured to reduce gel to gel variations.

Protein profiling using first, IEF on IPG strips and second SDS PAGE dimension separation, based on protein isoelectric point and size respectively, for 7, 11 and 24 cm format gel for superior resolution.

Differential gel electrophoresis (DIGE) 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.

2DE gels are scanned densitometricaly, analyzed using Image Master Platinum 7 software (GE Healthcare) for identifying statistical significance differentially expressed proteins.

Differentially expressed 2DE spots are excised digested with trypsin and identified by MALDI TOF TOF analysis.

Biomarker discovery and identification of proteins in immuno-precipitated samples and their interacting partners.

The protein sample isolation and quantification in SDS sample buffer, separated in short time on 1D SDS PAGE gel followed by commassie blue staining.

The whole protein stained band is cut and digested with trypsin.

The tryptic peptides are separated, analyzed on nano RP-UPLC connected to ESI QTOF mass spectrometry for MS and MSMS analysis.

The raw data in processed through PLGS search engine for protein identification and expression.

Gel Free Proteomics

  1. Absolute Quantification
  2. Labeled Quantification (Ex: iTRAQ)
  3. Label Free Quantification
  • Any body fluids
  • Extracted Animal, Microbe and plant proteome

All samples to be estimated for protein content before submission.

    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.
    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.
  • Isobaric 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.
  • These pooled samples are purified using HPLC (SCX column) then the fractions are run on LC-MSMS (ESI -QTOF) using Data Dependant Acquisition methodology for both Identification and Quantification.
  • The 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.
  • The iTRAQ runs performed in triplicates provides a robust statistics with optimized analysis.
  • 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.
A method in MS, which determines the relative amount of proteins in two or more biological samples based on their relative spectral intensity.
  • Two or more biological samples with different conditions e.g. Disease vs Treated or different environmental conditions run on LC-MS/MS (QTOF) instrument in triplicates using data independent acquisition methodology.
  • Peptides 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.
  • 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.
  • 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.
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