Abstract
The purpose of this study was to generate a basis for the decision of what protein quantities are reliable and find a way for accurate and precise protein quantification. To investigate this we have used thousands of peptide measurements to estimate variance and bias for quantification by iTRAQ (isobaric tags for relative and absolute quantification) mass spectrometry in complex human samples. A549 cell lysate was mixed in the proportions 2:2:1:1:2:2:1:1, fractionated by high resolution isoelectric focusing and liquid chromatography and analyzed by three mass spectrometry platforms; LTQ Orbitrap Velos, 4800 MALDI-TOF/TOF and 6530 Q-TOF. We have investigated how variance and bias in the iTRAQ reporter ions data are affected by common experimental variables such as sample amount, sample fractionation, fragmentation energy, and instrument platform. Based on this, we have suggested a concept for experimental design and a methodology for protein quantification. By using duplicate samples in each run, each experiment is validated based on its internal experimental variation. The duplicates are used for calculating peptide weights, unique to the experiment, which is used in the protein quantification. By weighting the peptides depending on reporter ion intensity, we can decrease the relative error in quantification at the protein level and assign a total weight to each protein that reflects the protein quantitation confidence. We also demonstrate the usability of this methodology in a cancer cell line experiment as well as in a clinical data set of lung cancer tissue samples. In conclusion, we have in this study developed a methodology for improved protein quantification in shotgun proteomics and introduced a way to assess quantification for proteins with few peptides. The experimental design and developed algorithms decreased the relative protein quantification error in the analysis of complex biological samples.
Highlights
Recent developments in methods and instruments for mass spectrometry enable quantitative proteomics analysis of complex samples with good coverage [1,2,3,4]
Isotope-coded affinity tag [7], isobaric tags for relative and absolute quantification1 [8], and stable isotope labeling by amino acids in cell culture (SILAC) [9] are among the most commonly used labeling methods based on stable isotopes. iTRAQ allows for simultaneous relative quantification of up to eight samples within a single run
We suggest an experimental design where each labeling set includes duplicate samples, and we describe how these duplicates are used for calculating peptide weights that can be used in addressing the accuracy of protein quantities
Summary
Recent developments in methods and instruments for mass spectrometry enable quantitative proteomics analysis of complex samples with good coverage [1,2,3,4]. Quantitative studies of complex human samples are subject to even more challenges related to large biological variation, large and unknown complexity of the human proteome and a large concentration range of proteins This in turn results in many peptides and a large variety of peptides that can cause interference and related problems in the mass spectrometry analysis. The quality of the protein quantifications is compared among several different mass spectrometers in this work; the influence of different loaded peptide amounts and the use of different methods for sample separation are examined Factors such as variance and bias of peptide quantification by iTRAQ are systematically evaluated in those high complex samples. Methods for improving the protein quantification are investigated; by filtering on the peptide level to remove low quality intensities and by weighting the peptide values to account for the higher risk of errors at low intensities [20]
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