JOURNAL OF PROTEOMICS 73 (2010) 769– 777 available at www.sciencedirect.com www.elsevier.com/locate/jprot Combining low- and high-energy tandem mass spectra for optimized peptide quantification with isobaric tags Loïc Dayona, Carla Pasquarellob, Christine Hooglandc, Jean-Charles Sancheza,b, Alexander Scherlb,⁎ aBiomedical Proteomics Group, Department of Structural Biology and Bioinformatics, Faculty of Medicine, University of Geneva, Geneva, Switzerland bProteomics Core Facility, Faculty of Medicine, University of Geneva, Geneva, Switzerland cProteome Informatics Group, Swiss Institute of Bioinformatics, Geneva, Switzerland ARTICLE INFO ABSTRACT Article history: Isobaric tagging, via TMT or iTRAQ, is widely used in quantitative proteomics. To date, Received 31 August 2009 tandem mass spectrometric analysis of isobarically-labeled peptides with hybrid ion trap– Accepted 31 October 2009 orbitrap (LTQ-OT) instruments has been mainly carried out with higher-energy C-trap dissociation (HCD) or pulsed q dissociation (PQD). HCD provides good fragmentation of the Keywords: reporter-ions, but peptide sequence-ion recovery is generally poor compared to collision- Quantitation induced dissociation (CID). Herein, we describe an approach where CID and HCD spectra are ESI combined. The approach ensures efficiently both identification and relative quantification iTRAQ of proteins. Tandem mass tags (TMTs) were used to label digests of human plasma and LC- Mass spectrometry MS/MS was performed with an LTQ-OT instrument. Different HCD collision energies were Orbitrap tested. The benefits to use CID and HCD with respect to HCD alone were demonstrated in Proteomics terms of number of identifications, subsequent number of quantifiable proteins, and Tandem mass tags quantification accuracy. A program was developed to merge the peptide sequence-ion m/z TMT range from CID spectra and the reporter-ion m/z range from HCD spectra, and alternatively to separate both spectral data into different files. As parallel CID in the LTQ almost doesn't affect the analysis duty cycle, the procedure should become a standard for quantitative analyses of proteins with isobaric tagging using LTQ-OT instruments. © 2009 Elsevier B.V. All rights reserved. 1. Introduction level. Labeling peptides after proteolytic digestion is mainly performed with isobaric tags such as tandem mass tags (TMTs) Isotopic dilution combined with mass spectrometry (MS) has or iTRAQ [5–7]. These isobaric compounds are synthesized with become the method of choice for discovery-based proteomics. heavy and light isotopes to have the same total mass, but to give Different strategies are used to introduce an isotopic label into a rise to reporter-ions at different masses after activation and complex biological sample. Typically, the label can be intro- subsequent tandem mass spectrometry (MS/MS or MS2). duced during cell growth [1–3] or during the sample preparation Activation is generally done using collision-induced dissocia- at the protein level (as, for example, ICAT [4]) or at the peptide tion (CID). Relative quantification between protein content of ⁎ Corresponding author. Proteomics Core Facility, Centre Medical Universitaire, Rue Michel-Servet 1, 1211 Genève 4, Switzerland. Tel.: +41 22 379 5494; fax: +41 22 379 5926. E-mail address: [email protected] (A. Scherl). 1874-3919/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jprot.2009.10.015 770 JOURNAL OF PROTEOMICS 73 (2010) 769– 777 different samples is then performed according to the ratio of In this report, we demonstrate that combining CID and HCD the reporter-ion abundances in the MS2 spectrum. activation modes in a linear ion trap–orbitrap (LTQ-OT) hybrid Isobaric tags have two major advantages compared to instrument allows precise peptide quantification without quantification in the first stage of mass spectrometry (MS1). compromising peptide identification with the linear ion trap First, this mode of operation provides an increased signal analyzer. To do so, a higher-energy HCD spectrum and a low to noise ratio of reporter-ions used for quantification due to energy CID spectrum were acquired on each selected precursor the removal of chemical noise in the second stage of mass ion. The CID spectrum was then used for peptide identification spectrometry. Second, all differentially labeled precursor ions at highest sensitivity, and the HCD spectrum was used for have the same mass. Thus, the MS1 spectrum is less complex, precise peptide quantification. TMTs were used as isobaric improving data-dependent precursor ion selection for in- labeling reagents. Different HCD collision energies were evalu- depth characterization of the sample. On the other hand, ated and their impact on peptide/protein identification and relatively high collision energy has to be applied in order to quantification was carefully assessed. A program, accessible obtain good reporter-ion statistics. However, higher collision over the internet, and compatible with all database search tools energy is not suitable to obtain peptidic backbone ions used to for the creation of composite spectra containing both qualitative match experimental MS2 spectra to peptides from protein and quantitative information was developed. Because this sequence databases. In addition, the relatively low reporter- program can also dissociate CID and HCD spectra, it can be ion masses (114 to 117 Da for iTRAQ and 126 to 131 for TMT) used for different data analysis schemes, as for example a are not suited for ion trap instruments. Indeed, these low- separated analysis pipeline for CID and HCD spectra. Finally, we mass ions are not stable during the activation step, a principle showed how this combined acquisition mode affected the known as the “1/3rd rule” [8]. instrument duty cycle and the number of quantifiable peptides. Recently, hybrid instruments combining the high sensitivity and versatility of linear ion traps with high performance orbitrap (OT) analyzers became available [9,10]. They have 2. Experimental section rapidly become popular for proteomics experiments. Basically, two activation methods using CID can be used with hybrid OT 2.1. Materials instruments for the analysis of low-mass fragment ions such as isobaric tag reporter-ions. The so-called “pulsed q dissociation” Human plasma, iodoacetamide (IAA, ≥99%) and tris(2- or PQD [11,12] consists of activating the precursor ion for a very carboxyethyl) phosphine hydrochloride (TCEP) were purchased short time at a high q value, where q is the Mathieu parameter, from Sigma (St. Louis, MO, USA). Triethylammonium hydrogen proportional to the applied radiofrequency voltage in the ion carbonate buffer (TEAB) 1 M pH=8.5 and sodium dodecyl trap. The q value is then lowered before the dissociation of the sulphate (SDS, ≥98%) were from Fluka (Buchs, Switzerland). precursor ion, allowing low-mass ions to be trapped as well. Formic acid (FA, 99%) was from Biosolve (Valkenswaard, the However, the sensitivity of this mode of operation is low Netherlands). Hydroxylamine solution 50 wt.% in H2O(99.999%) compared to traditional CID [13]. Also, the relatively low was from Aldrich (Milwaukee, WI, USA). Hydrochloric acid resolution of the ion trap does not allow precise quantification (25%) was from Merck (Darmstadt, Germany). Water for on closely separated ions such as isobaric tag reporter-ions. chromatography LiChrosolv® and acetonitrile Chromasolv® Higher-energy C-trap dissociation (HCD) is the second activation for HPLC (≥99.9%) were respectively from Merck and Sigma- method for the analysis of low-mass fragment ions. Activation Aldrich (Büchs, Switzerland). The duplex Tandem Mass Tags® and dissociation take place between the C-trap, used for the ion (TMT®) were kindly provided by Proteome Sciences (Frankfurt storage between the linear ion trap and the OT analyzer, and a am Main, Germany) and can be purchased from Thermo supplementary hexapole placed after the C-trap [14].This Scientific (Rockford, IL, USA). Sequencing grade modified fragmentation mode produces higher collision energy that trypsin was from Promega (Madison, WI, USA). efficiently generates the reporter fragments for quantification. However, this higher collision energy is associated with a 2.2. Sample preparation decreasing sensitivity due to ion scattering and a lack of backbone fragments useful for peptide identification [15]. Ten µL of plasma was dissolved in 990 µL of TEAB 100 mM Optimized quantitative analysis of phosphopeptides with adjusted to pH=8 with diluted HCl. To 200 µL of this solution, iTRAQ labeling has been recently reported and it has been 2 µL of SDS 1% and 4 µL TCEP 50 mM were added. The shown that classical CID yielded better phosphopeptide reduction was carried out at 60 °C for 1 h. Alkylation was sequence identification compared to HCD [16]. The authors performed by adding 2 µL of IAA 400 mM during 30 min in the − proposed to combine both modes for the quantitative analysis dark. Twenty µL trypsin 0.2 µg µL 1 (freshly prepared in the of phosphopeptides with iTRAQ. In the same way, this strategy above-mentioned TEAB solution) was added and the digestion has been applied very recently to study the changes in protein was proceeded overnight at 37 °C. The sample was divided expression in mouse hearts upon transverse aortic constric- into two identical aliquots. Duplex TMT labeling was achieved tion with CID followed by HCD of the precursor ions [17]. Better for 1 h, after addition of 40.3 µL of TMT duplex reagent in −6 performance with a CID/HCD hybrid acquisition strategy CH3CN (i.e., 0.83 mg, 2.42×10 mol). One plasma aliquot was was demonstrated relative to PQD acquisition. The authors labeled with TMT with reporter-ion at m/z=126.1 and the demonstrated the superior sensitivity and limit of quantifica- other with TMT with reporter-ion at m/z=127.1. Then, 8 µL of tion on digested bovine serum albumin (BSA) and applied their hydroxylamine 5% was added in each tube for reaction during quantification strategy to a complex protein mixture. 15 min. The differentially TMT-labeled samples were pooled JOURNAL OF PROTEOMICS 73 (2010) 769– 777 771 in a new tube and the resulting mixture was dried.