Evaluation and Improvement of Quantification Accuracy in Isobaric

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Evaluation and improvement of quantification accuracy in isobaric mass tag based protein quantification experiments Erik Ahrné, Timo Glatter, Cristina Viganò, Conrad von Schubert, Erich A. Nigg, and Alexander Schmidt J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.6b00066 • Publication Date (Web): 27 Jun 2016 Downloaded from http://pubs.acs.org on June 27, 2016

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Journal of Proteome Research

Evaluation and improvement of quantification accuracy in isobaric mass tag based protein quantification experiments

Erik Ahrné*, Timo Glatter, Cristina Viganò, Conrad von Schubert, Erich A. Nigg and Alexander Schmidt*

Biozentrum, University of Basel, Klingelbergstrasse 50/70, 4056 Basel, Switzerland

* Corresponding Authors Alexander Schmidt ([email protected], +41612672059) Erik Ahrné ([email protected], +41612672067)

Keywords Quantitative mass spectrometry, TMT, iTRAQ, label-free quantification

Abbreviations MS - Mass Spectrometry TMT - Tandem Mass Tags iTRAQ - Isobaric Tag for Relative and Absolute Quantitation SILAC - Stable isotope labeling by amino acids in cell culture LFQ - Label Free Quantification MS3 - Triple-stage mass spectrometry FDR - False Discovery Rate PSM – Peptide Spectrum Match SDC - Sodium deoxycholate

ACS Paragon Plus Environment

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Abstract

The multiplexing capabilities of isobaric mass tag based protein quantification, such as Tandem Mass Tags or Isobaric Tag for Relative and Absolute Quantitation have dramatically increased the scope of Mass Spectrometry based proteomics studies. Not only does the technology allow for the simultaneous quantification of multiple samples in a single MS injection, but its seamless compatibility with extensive sample pre-fractionation methods allows for comprehensive studies of complex proteomes. However, reporter ion based quantification has often been criticized for limited quantification accuracy due to interference from co-eluting peptides and peptide fragments. In this study, we investigate the extent of this problem and propose an effective and easy-toimplement remedy that relies on spiking a 6-protein calibration mixture to the samples. We evaluated our ratio adjustment approach using two large scale TMT 10-plex datasets derived from a human cancer and non-cancer cell line as well as E. coli cells grown at two different conditions. Furthermore, we analyzed a complex 2-proteome artificial sample mixture and investigated the precision of TMT and precursor ion intensity based Label Free Quantification. Studying the protein set identified by both methods, we found that differentially abundant proteins were assigned dramatically higher statistical significance when quantified using TMT. Data are available via ProteomeXchange with identifier PXD003346.

INTRODUCTION

State-of-the-art mass spectrometry based high-throughput shotgun proteomics allows for comprehensive study of proteomes of complex organisms. In several studies, more than 10,000 proteins have been identified from a single cell line1-4. However, to achieve this level of proteome coverage extensive pre-fractionation is required5,6, multiplying the amount of instrument time required to analyze a single sample. Since relative quantification of several perturbation states or time course analysis is often the objective of proteomics studies, the required instrument time multiplies again. To accommodate a sound statistical validation


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of differentially abundant proteins across the different samples, biological and technical replicates should be added, and this further increases the complexity of such mass spectrometry experiments. The multiplexing capabilities of isobaric chemical tag based quantification, such as TMT7,8 or iTRAQ9, supporting the analysis of up to 10 samples per MS injection, has therefore dramatically increased the scope of quantitative proteomics. Recently, hyperplexing workflows have been proposed combing iTRAQ or TMT with Stable Isotope Labeling by Amino acids in Cell culture (SILAC), allowing up to 30 samples to be quantified simultaneously10.

While peptide labeling with isobaric mass tags enables sensitive and precise multiplexed peptide and protein quantification, the accuracy of reporter ion based quantification is reduced due to interference from co-eluting and cofragmenting peptides11 and peptide fragments12. Important and systematic ratio compression has been reported for iTRAQ and TMT labeling experiments and several methods, both bioinformatic and experimental, have been proposed to minimize ratio distortion. This includes an algorithm, which detects co-isolated peptide signals in the MS1 spectrum and subsequently estimates and adjusts for co-fragmentation in the MS2 spectrum13. In another study, extensive fractionation, narrowing the precursor ion isolation width as well as delaying peptide fragmentation to occur closer to the apex of the chromatographic peak, have been demonstrated to lessen co-fragmentation and ratio compression14. Most success in reducing reporter ion interference has been demonstrated when combing isobaric labeling quantification with triple-stage mass spectrometry (MS3) and the multi-notch technology, but at the expense of significantly lower peptide and protein identification rates12,15.

In this study, we assessed the accuracy and precision of TMT based quantification by analyzing a complex two-proteome mixture. These test samples were also analyzed by MS1-level Label Free Quantification (LFQ), and thus allowing us to thoroughly compare these two popular quantification strategies. Next, we addressed the ratio distortion problem by investigating a novel approach relying on a 6-protein calibration mixture, spiked to the target


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samples. Finally, we propose a straightforward bioinformatics method to exploit the calibration data and improve the overall quantification accuracy. The merits of our ratio adjustment approach were demonstrated on two large-scale TMT 10-plex dataset; data acquired from human cancer cell lines and E. coli samples. Here, the adjusted quantification results were compared to those of the LFQ experiments as well as the quantification obtained when implementing an alternative previously published TMT/iTRAQ ratio correction method; the Signal-to-interference (S2I) correction method13.

EXPERIMENTAL SECTION Preparation of dilution samples

Experimental details describing cell culture and preparation of the different whole cell digests are detailed in the Supporting Information.

2-Proteome-Accuracy peptide samples

Dried peptides obtained from the whole cell digests of HeLa S3 or B. henselae were dissolved in an aqueous solution of 0.15% formic acid at a concentration of 0.5 µg/µl. To test the accuracy of TMT quantification, six peptide mixtures were prepared; six samples containing 0.5, 1.25, 2.5, 2.5, 3.75 and 25 µl of B. henselae peptide sample mixed with 40 µl of HeLa S3 peptide sample (see Table S2 for details). All samples were dried under vacuum and stored at -80°C until further processing. Next peptides were prepared and labeled with the individual 6-plex TMT labeling reagents as described below. Peptides were desalted on C18 reversed phase spin columns according to the manufacturer’s instructions (Microspin, Harvard Apparatus), dried under vacuum and dissolved at 0.5 µg/µl in aqueous solution of 0.15% formic acid.

The samples analyzed using label-free quantification included 50 µl of HeLa peptide sample mixed with 1, 1.5, 2 and 10 µl of B. henselae peptide sample. Finally, an aqueous solution of 0.15% formic acid was added to each mixture to


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reach a final volume of 100 µl (see Table S2 for details). Then, 4 µl of each peptide mix were employed for LC-MS analysis.

2-Proteome-Precision peptide samples

Dried peptides obtained from the whole cell HeLa S3 and B. henselae digests were both dissolved in an aqueous solution of 0.15% formic acid, each at a concentration of 0.5 µg/µl. To test the precision of TMT quantification, the following three peptide mixtures were prepared; 150, 150 and 300 µl of HeLa S3 sample mixed with 22.5, 30 and 30 µl of B. henselae peptide sample, respectively. Of each peptide mixture, aliquots containing 25 µg of human peptides were taken and dried under vacuum and stored at -80°C until further processing.

Calibration mixture

The

following

six

proteins;

PYGM_RABIT,P00489),

Phosphorylase

Conalbumin

B

(Uniprot

indentifiers:

(TRFE_CHICK,P02789),

Ovalbumin

(OVAL_CHICK,P01012), Beta-Casein (CASB_BOVIN,P02666), Beta-Galactosidase B

(GAL_ECOLI,P00722),

Alpha-lactalbumin

(B6V3I5_BOVIN,B6V3I5)

were

dissolved in lysis buffer (8 M urea, 0.1 M ammoniumbicarbonate) at a concentration of 2.5 mg/ml each, using strong ultra-sonication (two cycles of sonication for 20 seconds, Hielscher Ultrasonicator).

Different aliquots of each protein solution were mixed to produce six calibration mixtures. These contained all six standard proteins in different concentrations and generated a range of reference ratios, namely 0.25, 0.5, 2 and 4. Then, 1 µl of each calibration mix was spiked into the six peptide samples to be employed for TMT quantification, just before adding the TMT labeling reagent.

The exact volumes and concentrations of all solutions and peptides samples employed for preparing the different peptide dilution samples are illustrated in Table S2.


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TMT labeling Sample aliquots comprising 25 µg of peptides were labeled with isobaric tandem mass tags (TMT 6-plex or TMT 10-plex, Thermo Fisher Scientific) using a previously published protocol16 with a few modifications. Specifically, each of the TMT reagents was dissolved in 21 µl of DMSO, and 5 µl of each TMT reagent was added to the individual peptide samples solubilized in 20 µl labeling buffer (2 M urea, 0.2 M HEPES, pH 8.3). It is important to note that the dissolved reagents should be stored at -80°C and used within a few weeks after solubilization to maintain good labeling efficiency. After tagging peptides for 1 hour at 25°C, reactions were quenched by adding 1.5 µl of an aqueous 1.5 M hydroxylamine solution and incubating for another 10 minutes. After pooling all labeled peptide samples, the pH of the sample pool was increased to 11.9 by adding 1 M phosphate buffer (pH 12) and incubated for 20 minutes to remove TMT labels linked to peptide hydroxyl groups; these form during the labeling process as unwanted side products. Subsequently, the reactions were stopped by adding 2 M hydrochloric acid until a pH

Evaluation and Improvement of Quantification Accuracy in Isobaric

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