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Technical Note
Scout-MRM: multiplexed targeted mass spectrometry-based assay without retention time scheduling exemplified by Dickeya dadantii proteomic analysis during plant infection Blandine Rougemont, Sébastien Bontemps Gallo, Sophie Ayciriex, Romain Carriere, Hubert Hondermarck, Jean-Marie Lacroix, J.C. Yves Le Blanc, and Jerome Lemoine Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b03201 • Publication Date (Web): 27 Dec 2016 Downloaded from http://pubs.acs.org on December 28, 2016
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Analytical Chemistry
Scout-MRM: multiplexed targeted mass spectrometry-based assay without retention time scheduling exemplified by Dickeya dadantii proteomic analysis during plant infection
Blandine Rougemont1, Sébastien Bontemps Gallo2,5, Sophie Ayciriex1, Romain Carrière1, Hubert Hondermarck3, Jean Marie Lacroix2, J.C. Yves Le Blanc4, and Jérôme Lemoine1,*
1
Université de Lyon, Université Claude Bernard Lyon 1, Ecole Normale Supérieure de Lyon, Institut des Sciences Analytiques, CNRS UMR 5280, 5 rue de la Doua, F69100 Villeurbanne, France. Université de Lille, CNRS, UMR 8576 Unité de Glycobiologie Structurale et Fonctionnelle, F 59000 Lille, France.
2
School of Biomedical Sciences & Pharmacy and Hunter Medical Research Institute, University of Newcastle, Callaghan, NSW 2308, Australia.
3
4
Sciex, 71 Four Valley Drive, Concord, Ontario L4K 4V8, Canada.
5 Present
address: Laboratory of Zoonotic Pathogens, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT, USA
*Correspondence
should be addressed to J.L. (
[email protected]).
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Abstract
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Targeted-mass spectrometry of surrogate peptide panel is a powerful method to
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study the dynamics of protein networks, but chromatographic time scheduling
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remains a major limitation for dissemination and implementation of robust and
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large multiplexed assays. We unveil a Multiple Reaction Monitoring method (Scout-
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MRM) where the use of spiked scout peptides triggers complex transition lists
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regardless of the retention time of targeted surrogate peptides. The interest of
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Scout-MRM method regarding the retention time independency, multiplexing
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capability, reproducibility and putative interest in facilitating method transfer was
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illustrated by a 782-peptide-plex relative assay targeting 445 proteins of the
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phytopathogen Dickeya dadantii during plant infection.
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Analytical Chemistry
Introduction
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The proteomics toolbox is composed of distinct mass spectrometry-based
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strategies to embrace either the full horizon of protein expression or only a reduced
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landscape of regulated networks. Data Dependent Acquisition (DDA), also termed
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“shot gun” proteomics, still holds a lead position because its optimal analytical and
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computational workflows give rise to large protein data sets 1. More recently,
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peptide-centric orientated Data Independent Acquisition (DIA) has gained
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considerable attractiveness due to better quantification metrics, while keeping
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exhaustive sampling 2,3. No matter the pros and cons of DDA and DIA, both remain
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sophisticated approaches only mastered by specialized laboratories.
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Thanks to a simplified downstream bioinformatics, targeted dependent
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acquisition (TDA), namely Selected/Multiple Reaction Monitoring (SRM/MRM) 4–6
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and Parallel Reaction Monitoring (PRM)
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technology for sensitive assay of finite lists of proteins. Being implemented on the
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workhorse
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unparalleled sensitivity, specificity, throughput and inter-laboratory reproducibility
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10–12.
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than DIA in detecting predefined sets of peptides at low concentrations
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putative peptide targets exceed a few tens of units, they are monitored only around
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their expected elution time in order to keep reasonable dwell time and duty cycle. In
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practice, the multiplexing capability is drastically constrained by the likelihood of
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unintended RT shifts, which imposes scheduling windows 5 to 10 fold (2- to 5- min)
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larger than the peak width to secure the detection. Better management of RT shifts
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has been proposed with the concept of relative retention time indexation
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the fly” RT recalibration16. Nevertheless, both methods still retain the canonical
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principle of time scheduling that inevitably imposes a limiting compromise between
triple
quadrupole
7–9
modes, is considered a fit for purpose
instrument,
TDA-based
assay
demonstrates
Furthermore, two recent studies suggested that TDA might be more effective
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When
or ”on
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a secured detection and the extent of multiplexing. Despite valuable on-line
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transition repositories and web-based tools
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this persistent dependency of TDA to RT is still one of the main limitations that has,
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until now, prevented the expected wide dissemination of ready-to-use proteomic
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assays.
13,17,18
to support method development,
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Materials and Methods
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Selection of scout-peptides
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The 19 scout peptides were selected among a first set containing 45 synthetic
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peptides, which were proteotypic of human and Dengue virus species and identified
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as “best flyer” candidates during other research projects. Basic Local Alignment
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Search Tool (BLAST) verified proteotypicity and specificity against Dickeya
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dadantii. The selection of the 19 peptides was performed according to their ability to
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divide all target peptides in equal groups all along the chromatogram. In addition, a
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MRM experiment was performed to exclude the presence of any interferences
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around the peptide retention time and to define the detection threshold. A stock
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solution containing all scout-peptides at 500 ng/mL was spiked in all samples
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before LC-MS/MS analysis, as well as in blank sample.
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Mass spectrometry analysis
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Scout-MRM method was performed on a 1290 HPLC system (Agilent Technologies)
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coupled with a QTRAP® 6500 system equipped with a Turbo V™ source (Sciex) with
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a modified Analyst 1.6.2® version (Beta-version). LC parameters (gradient, columns,
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used injection volume) are the same as described above. The mass spectrometer was
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initially tuned and calibrated using polypropylene glycol, (SCIEX, Foster City, CA,
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USA) according to manufacturer’s instructions. Q1 resolution was adjusted to 0.7 ±
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0.1 Th. Q3 was also set to unit resolution in SRM mode. MS analysis was carried out
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in positive ionization mode using an ion spray voltage of 5,500 V. The nebulizer and
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the curtain gas flows were respectively set at 70 and 50 psi using nitrogen. The
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Turbo VTM ion source was set at 550◦C with the auxiliary gas flow (nitrogen) set at
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60 psi. All samples (reference and 3 kinetics growth conditions) were injected in
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triplicates.
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For academic research, Scout-MRM provisional software patch is available on
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request from Sciex (please contact
[email protected]).
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Creation of Scout-MRM method: selection of targeted proteins, peptides
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and transitions
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Scout-MRM method was built within Skyline. Proteins and peptides were sorted
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using the Refine setup. Repeated peptides were removed. At least 2 peptides were
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kept for protein identification. For the MRM-scout assay, only the two best
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responding peptides per identified proteins were selected (peptide settings; library
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criteria). Peptides were rank-ordered based on their intensity measured in MS1
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experiment. The transition settings were used to export both the best precursor ions
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and the 3 best intense fragment ions per peptide from the library. The “auto select
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all matching transitions” box was checked, and filter criteria were set to extract only
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2 or 3-charged precursor ions (ion type = p) and 1-charged fragment ion (ion types
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= y and b), from the ion 1 to the last ion. Instrument parameters were selected to
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match with a MRM experiment performed on a Triple Quadrupole instrument
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(targeted m/z from 200 to 1,000, DP and CE with an automatic calculation). In the
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Library setup, box was checked to pick only 3 best intense product ions from filtered
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products ions.
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Export of targeted transition lists and concatenation with the scout
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library
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The Scout and Dickeya dadantii peptide libraries were exported from Skyline data
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as csv files. These two libraries were then merged in one unique excel file
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containing, (i) for Dickeya dadantii peptides, their corresponding protein, the 3
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selected transitions, and their retention time, (ii) for scout peptides, the 2 best
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responding transitions and their retention time. All peptides were sorted according
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to their retention time in order to bracket Dickeya dadantii peptides with scout
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peptides and to structure the transition groups. We fixed at 150 the maximal
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transition number per triggered group to ensure a minimal dwell time of 5 ms and a
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target scan time set to 1.2 second. Only 3 groups were deleted from non-significant
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regulated proteins. 111 proteins were identified by 1 peptide and 334 were identified
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by 2 peptides. By using a predefined template derived from the sSRM-advanced
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method, the transition list was directly imported in Analyst 1.6.2 beta version
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(Figure S1). The final Scout MRM assay monitored 445 proteins by 782 peptides
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with 2346 transitions triggered by 19 scout peptides.
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Scout MRM data analysis
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Scout MRM data were integrated in Skyline software. Correct peptide identification
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was verified by the co-elution of the 3 monitored transitions, the conserved ratio
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between the 3 transitions across the injection triplicates (ratio transition CV < 5%),
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and a summed 3 transition area > 2000 cps/min. Only the peptides for which the
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summed area CVs were below 20% were selected for subsequent relative
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quantification. The relative-abundance of peptides was measured by comparing the
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peptide ratio from the different kinetic growth data points to the 0h reference
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culture. The logs of protein ratios were established by calculating the median of logs
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Analytical Chemistry
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of peptide ratios. 37 proteins that were identified only in the liquid growth media
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sample were discarded.
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All other materials and methods regarding the bacterial growth and plant
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infection, the sample preparation and proteomic experiments, and the MRM
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targeted analysis of virulence factors not detected in the DDA data sets are
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described as supplementary information.
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Results and Discussion
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We propose a new method to free MRM from time scheduling and thus
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enable the simplification of large targeted assay development from in-house or
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publicly available proteomic discovery data set. To reach these goals, we introduced
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the detection of known peptides dispatched all along the chromatogram (i.e. scout
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peptides) that successively manage the monitoring of complex transition groups
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(Figure 1, Figure S1). Once the first scout peptide (scout1) is detected above a
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defined threshold, it triggers the monitoring of a first group of peptides (group1),
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independently of their respective RT. As soon as scout2 becomes detectable, the
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monitoring of group1 is switched off while the affiliated new transition group2 is
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monitored, and so on. Hence, an exhaustive detection and analysis remain effective,
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even when voluntary or random RT drifts occur (Figure S2).
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As a test run, we focused on the bacteria Dickeya dadantii, a necrotrophic
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pathogen of numerous plants that is responsible for major production losses in
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agriculture (Figure 2). We first conducted a DDA shotgun proteomic analysis on
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proteomes of Dickeya dadantii strain 3937 extracted at 0h- (LB medium growth) or
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after 48h-infection of chicory leaves (Figure 2A; Supplementary Materials and
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Methods). A total of 19 synthetic proteotypic best flyer scout peptides (Table S1)
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were concomitantly spiked in the trypsin hydrolysates of the bacterial proteome, to
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assign each identified Dickeya dadantii peptide to a transition group surrounded by
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two scouts. From the set of identified proteins (FDR 1%), 473 proteins were inferred
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with at least 2 peptides and thus deemed good candidates for inclusion in the assay
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(Table S2). Among the main gene effectors of virulence reported in the literature 19,
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a few proteins were not detected in our discovery data set. We thus assessed their
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potential detectability by designing a fit for purpose MRM experiment, which
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ultimately lead to the additional detection of SodA and OpgH proteins with 4 and 2
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peptides, respectively (Supplementary Materials and Methods).
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We configured a preliminary targeted assay by merging the two exported
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transition lists extracted from Dickeya dadantii and scout peptides Skyline
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libraries, and ranking them in function of their RT. 150 transitions was nearly the
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upper-end limit within a peptide group to keep a duty cycle of at least 1.2-s.
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Therefore, only one peptide was retained in the targeted assay for proteins whose
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peptides were eluted in overwhelmed zones of the chromatogram, and that were not
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significantly regulated between 0 and 48h (P> 0,05; -2
