Investigating Acquisition Performance on the Orbitrap Fusion When

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Technical Note

Investigating Acquisition Performance on the Orbitrap Fusion when using Tandem MS/MS/MS Scanning with Isobaric Tags Christopher S. Hughes, Victor Spicer, Oleg V. Krokhin, and Gregg B. Morin J. Proteome Res., Just Accepted Manuscript • Publication Date (Web): 18 Apr 2017 Downloaded from http://pubs.acs.org on April 18, 2017

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Journal of Proteome Research

Investigating Acquisition Performance on the Orbitrap Fusion when using Tandem MS/MS/MS Scanning with Isobaric Tags Christopher S Hughes1, Victor Spicer2, Oleg V Krokhin2,3, Gregg B Morin1,4,* 1 – Canada’s Michael Smith Genome Sciences Centre, British Columbia Cancer Agency, Vancouver, British Columbia, Canada 2 – Manitoba Centre for Proteomics and Systems Biology, University of Manitoba, Winnipeg, Manitoba, Canada 3 – Department of Internal Medicine, University of Manitoba, Winnipeg, Manitoba, Canada 4 – Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada

* To whom correspondence should be addressed: [email protected]

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Abstract Methods for isobaric-tagged peptide analysis (e.g. TMT, iTRAQ), such as the synchronous precursor selection (SPS) tandem MS/MS/MS (MS3) approach, enable maintenance of reporter ion accuracy and precision by reducing the ratio compression caused by co-isolated precursor ions. However, the decreased throughput of the MS3 approach necessitates careful optimization of acquisition strategies and methods to ensure maximal proteome coverage. In this study we present a systematic analysis of acquisition parameters used to analyze isobarictagged peptide samples on current generation Orbitrap mass spectrometer (MS) hardware. In contrast to previously reported works, we demonstrate the limited utility of acquiring reporter ion data in the ion trap analyzer; ion trap acquisition had only a minimal increase in identification depth and reduced quantification precision. We establish that despite the significantly increased scan rate afforded through the use of higher-energy collisional dissociation (HCD) in MS3-based ion trap isobaric tag analyses, the reduced quantification precision and reporter ion yields negate the potential benefits in proteome coverage. Lastly, using optimized parameter sets, we further demonstrate the limited utility of the ion trap detector versus the Orbitrap for reporter ion detection in an in-depth analysis of a complex proteome sample. Together, these data will serve as a valuable resource to researchers undertaking analysis on current generation Orbitrap instrumentation with isobaric tags.

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Keywords Tandem mass tagging, isobaric labeling, Orbitrap, MS/MS/MS analysis, quantitative proteomics, ion trap, HCD, CID, multi-notch isolation, synchronous precursor selection



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Introduction The ability to profile thousands of proteins in a quantitative manner on a rapid timescale has elevated mass spectrometry (MS) to be the tool of choice for proteome analysis. With this success has come the need to profile multiple conditions or samples in an efficient manner while balancing statistical power and throughput, resulting in the advent of methods designed to enable increased numbers of comparisons at a minimal cost of MS time 1. Of these, technologies based on isobaric mass tags (e.g. iTRAQ 2, TMT 3) offer the greatest flexibility due to their high level of multiplexing (4-8 plex for iTRAQ, 2-10 plex for TMT) and sample compatibility (e.g. no requirement for incorporation of a metabolic label, such as in SILAC 4). Each isobaric tag consists of a unique ‘reporter ion’ group coupled to a mass balancing region and utilizes NHS chemistry to specifically and efficiently tag primary amines present on the N-terminus and lysine sidechains of every peptide in a typical proteolytic digest. The resulting multi-sample peptide mixture can be analyzed in a single MS run, and the contributions of the original multiplexed samples to a given ion signal determined from the reporter ion intensities in a tandem MS/MS scan (MS2). Problematically, the compression of isobaric tag ratios stemming from coisolated precursor ion populations has been demonstrated to have a negative impact on quantification accuracy 5. To mitigate errors in quantification stemming from precursor isolation, methods involving additional instrument or postacquisition processing manipulation steps have been developed to increase the precision and accuracy of reporter ion data

6–12

. One method uses MS/MS/MS


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(MS3) acquisition of reporter ion intensities generated from higher-energycollisional dissociation (HCD) of a peptide fragment observed in an MS2 scan of a precursor population that has undergone collisional induced dissociation (CID) 8. This method can be carried out on current and previous generation Orbitrap MS instruments with minor method modifications 8. Newer implementations of this approach that utilize the synchronous selection of multiple precursors (SPS) from the MS2 scan to improve the sensitivity of the approach are available only on current generation MS instruments

13

, such as the Orbitrap Fusion

14

and Fusion

Lumos. Problematically, the SPS-MS3 method for reporter ion quantification necessitates the acquisition of an additional scan for every data-dependent triggered event, effectively halving the cycle time available for capturing MS2 data. As a result, maintaining the depth of identification and quantification coverage requires extended fractionation or analysis regimes 15. A recent technical study noted that peptide identification performance could be improved by manipulating the instrument acquisition pathway in SPSMS3 scan methods

16

. The authors demonstrated that after a CID-MS2 scan then

performing the subsequent HCD-MS3 acquisition in the ion trap (IT) instead of the slower Orbitrap (OT) detector they could obtain 91%, 84%, and 66% increases in peptide

spectral

matches

(PSM),

peptides,

and

proteins,

respectively.

Importantly, this increase in speed had minimal impact on reporter ion accuracy, as TMT tags detected in the IT yielded a percent error of just 5.5% compared with 3.8% in the OT. The caveat of this technique is that it can only be used with the TMT 2- or 6-plex reagents, as the ion trap cannot resolve the isotopologue 5 ACS Paragon Plus Environment


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masses of the individual TMT 10-plex tags

17,18

. While this work presented a

sufficient proof of concept for this scan methodology in isobaric tag experiments, only small sets of runs were performed and a limited number of scan modes were tested. A systematic examination of the IT and other operation and analysis modes of current generation MS hardware with respect to isobaric tags would be of significant benefit to those considering to, or actively utilizing these technologies. In this work we employ a standard, high-complexity Human cell line tryptic-digest combined with a synthetic peptide library to systematically evaluate the properties of a variety of MS operation modes and detector configurations for the optimal examination of isobaric-tagged peptide mixtures. We demonstrate that despite the increased MS3 scan rate of the IT, the benefit in peptide and protein identifications when examining complex mixtures is minor, while the negative impact on quantification precision is significant. We further demonstrate that the utilization of HCD in MS2 scans provides a significant increase in scan speed, but comes at the cost of ion yields in MS3 scans. Lastly, using an in-depth analysis of a fractionated Human cell line proteome we demonstrate that IT-MS3 scanning is also not capable of recovering the lost throughput that results from the inability to utilize 10-plex TMT tags. Together, these data highlight the characteristics of a variety of scan parameters and modes on the Orbitrap Fusion MS for the use of isobaric-tagged peptides to balance coverage and accuracy versus MS experiment time.


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Experimental Section General Reagents and Chemicals When possible, pre-made, nuclease-free versions of all solutions were used. Experimental solutions were prepared from stock solutions of: 1M HEPES pH

7.3,

nuclease-free

(Thermo

Fisher,

CAT#BP299-1),

tris(2-

carboxyethyl)phosphine hydrochloride (TCEP) (Sigma, CAT#C4706), 20% SDS (Thermo Fisher, CAT#BP1311-1), and nuclease-free water (Thermo Fisher, CAT#AM9937).

Chloroacetamide

(CAA)

was

obtained

from

Sigma

(CAT#C0267). For LC-MS experiments, water (CAT#51140), acetonitrile (CAT#51101), and formic acid (CAT#85178) were obtained from Thermo Fisher. Paramagnetic Beads Unless otherwise stated, all experiments used a 1:1 combination of two different types of carboxylate-functionalized beads, both with a hydrophilic surface (Sera-Mag Speed Beads, GE Life Sciences, CAT#45152105050350 and CAT#65152105050350). The magnetic particles are an average diameter of 1µm. The beads were stored at 4°C at a concentration of ~12 mg/mL. Magnetic racks used in all experiments were designed and manufactured in-house as described previously 19. Cell culture and harvest Cell pellets were obtained for the lines: Hs578t, HeLa-S3, U2-OS, 293T, HCT-116, K562, A549, HepG2, TOV-21G, PANC-1, PC3, Jurkat, and SK-MEL-2. Cells were grown and harvested by the National Cell Culture Center (Biovest International). A total of ~1e9 cells were grown for each line, and provided as 7 ACS Paragon Plus Environment


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aliquoted pellets at a concentration of ~5e7 cells per tube. Cells were stored at -80°C until use. Protein isolation, reduction, and alkylation Cell pellets (~5e7 cells each) from each of the above lines were prepared using the following protocol. Pellets were thawed on ice and periodically vortexed. To each pellet, 800µL of lysis buffer (100mM HEPES pH 7.3 and 2% SDS, 50mM NaCl, 10mM TCEP, 40mM CAA, 1X cOmplete protease inhibitor – EDTA free (Sigma, CAT#11836170001) was added, and the pellets vortex mixed. Lysis mixtures were passed through a 21-gauge needle mounted to a 1mL syringe (BD Biosciences) a total of 5 times and transferred to 2mL FastPrep-compatible tubes containing Lysing Matrix D (MP Biomedicals, CAT#116913050). Lysis mixtures were vortexed on the FastPrep-24 instrument (6 M/s, 45 seconds, 1 cycle). Tubes were centrifuged at 20,000g for 10 minutes, and the supernatant recovered. Resultant lysates were heated at 90°C for 10 minutes, and chilled to room temperature for a further 30 minutes. Protein concentrations were measured using the Pierce BCA Protein Assay Kit (Thermo Fisher, CAT#23225) on a NanoDrop spectrophotometer (Thermo Fisher). Protein clean-up with SP3, and protease digestion A single equal protein mass mixture of all protein isolates from the 13 original cell lines was prepared based on concentration values from a BCA assay. A total of ~1mg of protein was prepared in a final volume of 100µL. To the mixture, 20µL of each type of SeraMag Speed Bead was added, and proteins purified using the SP3 method 19. Briefly, to promote binding to the beads, 150µL 8 ACS Paragon Plus Environment

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of acetonitrile (HPLC grade, Thermo Fisher, CAT#A998-4) was added to achieve a final concentration of at least 50% and samples incubated for 10 minutes at room temperature. Tubes were placed on a magnetic rack and incubated for 2 minutes. The supernatant was discarded, and beads were rinsed 2X with 180µL of 70% ethanol, and 1X with 180µL of acetonitrile while on the magnetic rack (the beads were not resuspended). Tubes were removed from the magnetic rack, and beads resuspended in 100µL of 100mM HEPES, pH 7.3 containing 20µg of trypsin/rLysC mix (Promega, CAT#V5071) and incubated for 14 hours at 37°C in a PCR thermocycler. After incubation, the tubes were pipette mixed to resuspend the beads and 50µL of acetonitrile was added. Tubes were incubated for 10 minutes at room temperature, and placed on a magnetic rack for a further 2 minutes. The supernatants were recovered to fresh 1.5mL polypropylene micro-tubes. Design of synthetic peptide mix The set of standard peptides was designed to fulfill following criteria: i) peptides should be easily detectable; 8-16 amino acids in length; ii) should not contain residues prone to spontaneous chemical modifications (Met, Trp, Asn, Gln); iii) have no overlap with tryptic peptides derived from human proteins. These sequences were mined from 2D-LC-MS/MS (high pH RP – low pH RP) data previously acquired from whole cell digests of Yarrowia Lipolytica and Ralstonia Eutropha. The output identifications were filtered by retention time prediction in both LC dimensions

20

to remove false positive identifications,

yielding 81187 unique non-modified peptides in total. Experimental retention 9 ACS Paragon Plus Environment


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values for all species were accurately aligned (mapped) in both chromatographic dimensions using a set of peptide retention standards developed in our laboratory 21,22. These peptides were arranged into 24 equally sized bins across the first dimension (pH 10) of the separation space. A Monte Carlo method (500 iterations) was then applied to randomly select from this set a population of 1000 peptides with the objective of minimizing the correlation in observed retention times in the first and second separation dimensions (i.e. maximizing separation orthogonality). Selection also maintained the population distribution in the first dimension consistent with the total working set’s binning, ensuring that peptides found in each pH 10 fraction had wide distribution across the separation space in the second dimension (acidic pH with formic acid as the ion pairing modifier). A set of 550 unique sequences was selected and synthesized as a pooled SpikeMix (JPT Peptide Technologies, Table S-1). Tandem mass tag labeling of peptides TMT 6-plex labeling kits were obtained from Pierce. Each TMT label (5mg per vial) was reconstituted in 500µL of acetonitrile and refrozen. An equal amount (approximately 40µg) of the 13-cell-line peptide mixture was aliquoted to each of 6 fresh 1.5mL tubes. Labeling reactions were carried out through addition of 160 µg of TMT label in two volumetrically equal steps of 8µL (80 µg per addition), 30 minutes apart. Reactions were quenched through addition of 5µL of glycine (1M stock solution) (Sigma). Labeled peptides were concentrated on a SpeedVac centrifuge (Thermo Scientific) to remove excess acetonitrile, acidified to 1% (v/v) 10 ACS Paragon Plus Environment

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trifluoroacetic acid (TFA), combined at specific ratios as indicated, and purified with a C18 cartridge (50 mg C18-t, SepPak, Waters, CAT#WAT054960). Peptide clean-up procedures Peptides were desalted and concentrated using either SepPak or StageTip treatment. For SepPak clean-up, cartridges (50 mg C18-t, Waters, CAT#WAT054960) were rinsed twice with 0.6mL of acetonitrile with 0.1% TFA. Cartridges were then rinsed twice with 0.6mL of water with 0.1% TFA prior to sample loading. Loaded samples were rinsed three times with 0.1% formic acid (0.6mL per rinse) and eluted with 1.2mL of 80% acetonitrile containing 0.1% formic acid. StageTips were prepared as previously described 23. StageTips were rinsed and eluted using the same conditions as with SepPak cartridges. In both cases, eluted samples were concentrated in a SpeedVac centrifuge (Thermo Scientific) and subsequently reconstituted in 1% formic acid. High-pH Reversed Phase C18 Peptide fractionation High-pH reversed phase analysis was performed on an Agilent 1100 HPLC system equipped with a diode array detector (254, 260, and 280nm). Fractionation was performed on an XTerra C18 column (3.0 x 150mm, 3.5µm, Waters). Elution was performed at a flow rate of 0.3mL per minute using a gradient of mobile phase A (20mM ammonium hydroxide, pH 10) and B (acetonitrile). The gradient began at a baseline of 5% B for 10 minutes, to 14% B in 4 minutes, to 28% B in 16 minutes, to 40% B in 8 minutes, to 80% B in 5 minutes, held at 80% for 4 minutes, to 5% B in 1 minute, and a final reconditioning at 5% for 8 minutes (60 minutes total runtime). Fractions were 11 ACS Paragon Plus Environment


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collected every minute across the entire sample run and gradient length (48 total fractions) and concatenated into 12 final samples. Fractions were dried in a SpeedVac centrifuge and reconstituted in 1% formic acid prior to MS analysis. Mass Spectrometry Data Acquisition Analysis of TMT labeled peptide fractions was carried out on an Orbitrap Fusion Tribrid MS platform (Thermo Scientific) as described previously

15

with

modifications. Samples were introduced using an Easy-nLC 1000 system (Thermo Scientific). Columns used for trapping and analytical separations were packed in-house. Trapping columns were packed in 100µm internal diameter capillaries to a length of 30mm with C18 beads (Reprosil-Pur, Dr. Maisch, 5µm particle size). Trap columns were fritted in-house using a combination of formamide and Kasil (1:3 ratio). Trapping was carried out for a total volume of 20µL at a pressure of 300 bar. After trapping, gradient elution of peptides was performed on a C18 (Reprosil-Pur, Dr. Maisch, 5µm particle size) column packed in 75µm internal diameter capillaries with pulled and fritted nanospray tips (PicoFrit, 15µm tip, New Objective) to a length of 40cm and heated to 45°C using AgileSLEEVE column ovens (Analytical Sales & Service). Elution in 120-minute runs was performed with a gradient of mobile phase A (water and 0.1% formic acid) from 2 – 6% B (acetonitrile and 0.1% formic acid) over 5 minutes, 6 – 22% B over 80 minutes, and to 40% B over 15 minutes, with final elution (80% B) and equilibration (2% B) using a further 20 minutes at a flow rate of 350nL/min. Elution in 60-minute runs was performed with a gradient of mobile phase A (water and 0.1% formic acid) from 2 – 6% B (acetonitrile and 0.1% formic acid) 12 ACS Paragon Plus Environment

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over 5 minutes, 6 – 22% B over 27 minutes, and to 40% B over 8 minutes, with final elution (80% B) and equilibration (2% B) using a further 20 minutes at a flow rate of 350nL/min. Data acquisition on the Orbitrap Fusion (control software version 2.1.1565.20) was carried out using a data-dependent method with multi-notch synchronous precursor selection MS3 scanning for TMT tags. The Orbitrap Fusion was operated with a positive ion spray voltage of 2100 and a transfer tube temperature of 275°C. Since a wide range of method settings were tested in this work, each set of parameters will be referred to with an index number throughout the text that can be referenced to obtain the complete method information (Table S-2). In addition, all acquisition methods are available in their native format as indicated in the ‘Data and Code Availability’ section. Mass Spectrometry Data Analysis Data from the Orbitrap Fusion were processed using Proteome Discoverer Software (ver. 2.1.1.21). MS2 spectra were searched using Sequest HT against a combined UniProt Human proteome database appended to a list of common contaminants (24,624 total sequences). Sequest HT parameters were specified as: trypsin enzyme, 2 missed cleavages allowed, minimum peptide length of 6, precursor mass tolerance of 50ppm, and a fragment mass tolerance of 0.6 (IT) or 0.05 (OT) Daltons. Oxidation of methionine, and TMT at lysine and peptide Ntermini were set as variable modifications. Carbamidomethylation of cysteine was set as a fixed modification. Peptide spectral match error rates were determined using the target-decoy strategy coupled to Percolator modeling of positive and 13 ACS Paragon Plus Environment


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false matches

24,25

. Data were filtered at the peptide spectral match-level to

control for false discoveries using a q-value cut off of 0.01 as determined by Percolator. Data sets generated in Proteome Discoverer were exported and analyzed with a combination of scripts built in R designed in-house. Contaminant and decoy proteins were removed from all data sets prior to downstream analysis. Bioinformatic and Statistical Analysis General statistical parameters In all boxplots, center lines in plotted boxes indicate the median, upper and lower the 75th and 25th percentiles, and upper and lower whiskers 1.5X the interquartile range. All correlation calculations utilize the Pearson method. The calculation of individual p-values was performed using two-sided, two-sample Student’s t-tests assuming an equal variance, unless otherwise indicated. Reporter Ion Quantification Reporter ion values were calculated using Proteome Discoverer (ver. 2.1.1.21). Values for the reporter ions were calculated as intensities to ease the comparison of IT and OT data. Reporter ions were quantified from MS3 scans using an integration tolerance of 500 ppm (IT) and 50 ppm (OT) with the most confident centroid setting. Reporter ion values were corrected for isotopic impurities using the manufacturer provided factors. Variances of the TMT reporter ions from the expected ratios were calculated at the peptide level. For each peptide, the total intensity was 14 ACS Paragon Plus Environment

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calculated and used to determine the fractional intensities for each TMT channel. The resultant values were then multiplied by the number of equivalents of sample in each TMT channel (e.g. 3:3:3:1:1:1 = multiply by 12). In all calculations, percent differences were determined as ((x – y) / y) * 100 (see Table S-3 for scripts). Data and Code Availability The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium dataset

identifier

26

via the PRIDE

PXD005890

(Username:

27

partner repository with the [email protected],

Password: KrFz4mAT). Identification results, MS acquisition methods, and sequence databases are also stored on ProteomeXChange under the same identifier. Descriptions of the stored scripts used to process the data can be found in Table S-3. All scripts can also be accessed openly in GitHub at: https://github.com/chrishuges/tmt_ratePaper.



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Results and Discussion The goal of this work was to test and validate a suitable set of parameters that enabled efficient and accurate analysis of isobaric tag labeled peptide mixtures using current generation MS hardware. We define efficiency in this context as maximizing identification sensitivity (e.g. fast scan acquisition rate, maximal number of peptide identification events) without sacrificing data quality (e.g. quantification precision). Although the methodologies used in previous studies have enabled in-depth identification and quantification of the proteome, we hypothesized that the overall efficiency of the analysis (e.g. equivalent or greater proteome coverage in less time with equivalent or greater quantification precision) could be potentially improved by further optimization of the MS acquisition parameters

15

. To test a wide variety of scan and parameter settings,

a standard mixture of cell lines and synthetic peptides was prepared. The standard is a complex tryptic digest of a combined pool of 13 individual Human cell lines labeled with TMT 6-plex reagents (1:1:1:1:1:1, TMT126 – TMT131). To this mixture, a set of 550 synthetic peptides labeled with TMT 6-plex reagents (3:3:3:1:1:1, TMT126 – TMT131) was added. An on-column injection of 1µg of total peptide derived from the cell line and synthetic peptide mixture was used for all analyses in 120-minute runs in technical triplicate (n = 3). The use of a single, complex, isobaric-labeled peptide mixture in all experiments provided a suitable dataset for direct comparison of all tested parameters and scan modes.


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Coverage and accuracy of peptide identifications when using the OT versus IT in MS3 scanning To determine the impact on peptide identification and quantification when using MS3 with OT (OT-MS3) and IT (IT-MS3) analyzers, we compared these scan modes to the cell line standard analyzed with conventional MS2-only (OTMS2) scanning (methods A1 – A3, Table S-2). Using the mean of triplicate injections, we observed a decrease of 22%, 32%, and 19% in the numbers of MS2 spectra, unique peptides, and protein identifications when using a standard OT-MS3 method compared with OT-MS2 (Table 1). Using the IT-MS3 scanning approach, we observed an 8% increase in the number of acquired MS2 spectra, validating the improved performance in scan rate with the IT

16

. However, this

increase did not translate to improved identification metrics, with the IT-MS3 approach identifying 21% and 12% fewer unique peptides and proteins compared with OT-MS2 (Table 1); this may stem from the improved HCD fragmentation of TMT labeled peptides.

In the quantification data, we observed that the OT-MS3 method averaged 0.11 missing reporter ion values per MS3 across all acquired spectra, compared with 0.060 in the standard OT-MS2 analysis (p = 4.8e-4, Students t-test). In the IT-MS3 data, an average of 1.7 reporter ion values were missing per MS3 across 17 ACS Paragon Plus Environment


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all acquired spectra (p = 1.7e-7, Students t-test). To measure quantification precision, the data were filtered to retain only peptides from the synthetic spike that were reported with a maximum of 1 missing value per spectra. Surprisingly, despite the potential impact of peptide co-isolation, we observed similar errors in quantification comparing OT-MS2 (mean across all channels = 20%) with OTMS3 analysis (mean across all channels = 21%) (Figure S-1a, S-1b). However, in the IT-MS3 data we observed higher errors in quantification compared with both the OT-MS2 and OT-MS3 methods, at 30% across all channels (Figure S-1c). Taken together, these data demonstrated that despite the rapid scan rate afforded by the use of the IT in MS3 analyses, there was not a significant improvement in peptide and protein identifications, and a negative impact on quantification precision. When we compared the identification rates between this work and that of Liu et al.

16

, we observed a more modest 15% increase in unique peptide

identifications with IT-MS3 compared to OT-MS3 (Table 1) (84% reported by Liu et al.). Examining the base-peak chromatograms from Liu et al (~250ng of peptide on-column) revealed that the cell line mixture utilized in this work (~1000ng of peptide on-column) was more complex across the chromatographic elution window (Figure S-2). The result of this differential complexity could be seen in the scan rates and fill time metrics of the data files from the Liu et al and this study. An average of 32,793 and 42,453 MS2 scans were obtained in OTMS3 and IT-MS3 runs across a 2-hour acquisition window in this study, compared with 18,794 and 26,289 in the same time frame in Liu et al. Investigating the ion 18 ACS Paragon Plus Environment

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injection times in the Liu et al files revealed that 9358/18800 OT-MS3 scans (average across 5 technical replicates) reached the maximum allowable fill time (49.8%, mean injection time across all runs = 173ms). In contrast, just 1542/32793 (5%, mean injection time = 47.5ms) of scans reached the maximum in this study across 3 technical replicates; despite using the same max fill time setting (250ms). We speculate that a combination of the slow overall scan rate and low complexity of the yeast digest in Liu et al led to an amplified bias against the OT analyzer. Effects of scan parameters for IT and OT on MS3 yield and accuracy To determine if any additional increases in acquisition rate could be achieved without sacrificing quantification accuracy for the Human cell line sample, we systematically tested a range of method configurations (Table 2, Table S-2). When using the standard SPS-MS3 method configuration (CID fragmentation for MS2 and HCD fragmentation for MS3) we further observed that the use of the IT analyzer for MS3 analysis provided increases in the numbers of MS2, unique peptides, and proteins compared with OT-MS3 (Table 2). When the MS3 scan rate was increased from Normal (33,333 Daltons/second) to Turbo (125,000 Daltons/second) in the IT (methods A3 – A5), the mean numbers of MS2, unique peptides, and proteins across triplicate measurements could be improved, without a significant impact on quantification precision of the synthetic spiked peptides (Table 2). However, compared to the OT-MS3 (0.11, p = 2.4e-8, Students t-test), IT-Normal (1.1, p = 8.3e-7, Students t-test), and IT-Rapid (1.7, p = 1.9e-6, Students t-test) acquisition approaches, the IT-Turbo method had a



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mean of 3.0 missing values per MS3 spectrum across all scans. Therefore, despite improving the proteome coverage when compared with OT-MS3 methods, increasing the scan rate negatively impacted quantification in the ITMS3 methods.

In addition to the standard SPS-MS3 method configuration, HCD fragmentation can be substituted for CID to generate fragment ions for MS2 scans. Pairing the improved duty cycle of an HCD fragmentation and scan event with OT-MS3 detection (method A6) yielded a 7% increase in the number of MS2 spectra when compared with conventional CID-MS2 with OT-MS3 analysis (Table 2). However, this increase in MS2 spectra did not translate to a substantial difference in the numbers of unique peptides (0.04%) and proteins (-0.7%). When utilizing HCD-MS2 in combination with IT-MS3 at the ‘Normal’ scan rate to maximize the relative quality of the quantification results (method A7), 39%, 20%, and 10% increases in the numbers of MS2, unique peptides, and proteins was observed compared to conventional OT-MS3 analysis (Table 2). Surprisingly, the improvements when pairing HCD-MS2 with the IT-MS3 ‘Normal’ scan rate are comparable to those when using CID-MS2 with IT-MS3 in the ‘Turbo’ mode (Table 2). However, despite increases in scan rate and identification depth offered with


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the HCD and IT operation modes, the conventional CID with OT-MS3 approach yielded the greatest precision in quantification.

To further investigate the limited difference in identification depth with the more rapid HCD prior to MS2 when compared to the conventional approach (CIDMS2 – OT-MS3), we examined fragmentation properties during these acquisitions (methods A8 – A17). The increased chance for secondary fragmentation events can potentially limit the utility of HCD in MS2 for quantification in MS3 analyses, as the TMT reporter ions may be lost. In ramping the HCD-MS2 normalized collision energy (NCE) values from 25 – 40, we observed that an NCE of 35 provided the optimum number of unique peptide identifications (Table 3). However, measuring the mean of the reporter ion signal across all channels and peptides in triplicate measurements, the overall intensity drops significantly as the energy is increased compared to the CID-MS2 methods (Table 3). This suggests that secondary fragmentation events with HCD are increased as the NCE is elevated, reducing the numbers of reporter ions available to contribute in a subsequent OT-MS3 scan. Although modulating the NCE values for the subsequent OT-MS3 scan improved the reporter ion signal, the relative difference



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to the optimum CID-MS2 approach remains above 20% (Table 4). Together, these data indicate that despite the improvement in scan rate and identification depth with HCD-MS2, the accuracy and overall reporter ion yield in quantification suffered with this approach. OT-MS3 versus IT-MS3 for comprehensive proteome analysis To determine the capabilities of the optimized OT-MS3 and IT-MS3 scanning regimes in a comprehensive proteome analysis, the cell line mixture with the synthetic spikes was re-analyzed after prior off-line peptide fractionation (method A2 – A3). One of the main caveats of using the IT detector for MS3 acquisition is the requirement that a sufficient mass differential is present between the reporter tags such that they can be resolved. Given that TMT 10plex reagents afford an approximate 65% increase in throughput relative to their 6-plex counterparts, IT-MS3 methods should achieve performance metrics that recover this loss that may justify their use in place of OT-MS3 workflows. To test this, the total analysis time for the OT-MS3 and IT-MS3 methods was scaled to approximately reflect this throughput difference (12 fractions x 2-hour runs each for OT-MS3, 12 fractions x 1-hour runs each for IT-MS3). Comparing the OT-MS3 and IT-MS3 analyses revealed that despite the rapid analysis rate of IT-MS3, the numbers of MS2 (-80%), unique peptides (-106%), and proteins (-22%) were all significantly decreased relative to OT-MS3 (Figure 1). Notwithstanding the differences in depth of coverage, the relative quantification observed with IT-MS3 displayed a similar accuracy (mean percent error: IT-MS3 = 23.0, OT-MS3 = 22.1; n = 443) but less precision (standard deviation: IT-MS3 = 17.8, OT-MS3 = 13.8; n


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= 443) when compared with OT-MS3 (r2 = 0.42, n = 443) for matching the expected log2 fold-change quantification of 1.6 (3:1) of the synthetic spiked peptides (Figure 1). When considered in combination with the significant decreases in multiplexing, depth of coverage, and increases in relative quantification error, the use of IT-MS3 in the place of OT-MS3 is of limited utility in any of the experimental setups tested in this work.

In this study we have performed an in-depth examination of acquisition method configurations and their related performance metrics in isobaric labeling experiments on an Orbitrap Fusion MS. We highlighted the limited utility of the IT detector for MS3 scans in SPS-MS3 experiments, in contrast to previous works. We further reveal a range of alternative method configurations that yield benefits in terms of proteome coverage, but sacrifice the overall quality of the quantification results in terms of precision and reporter ion yield. Together, these data represent a useful resource of validated method configurations and parameters that can be utilized in SPS-MS3 experiments utilizing isobaric tagging on current generation Orbitrap instruments.



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Supporting Information The following files are available free of charge at ACS website http://pubs.acs.org: •

Supporting legends for figures S-1 and S-2 relating to the measurement of quantification precision with different detectors, and data complexity. Supporting legends for tables S-1 – S-3 describing provided datasets.

•

Supporting tables containing synthetic peptide library sequences (S-1), acquisition method settings (S-2), and R-script details (S-3).

Acknowledgements C.S.H. would like to acknowledge valuable contributions, input, and discussions from Lida Radan. C.S.H. would also like to acknowledge Jane M. Liu for correspondence related to her previously published work.

Funding Sources This work was supported by the British Columbia Cancer Foundation (G.B.M., C.S.H).

Competing Financial Interests The authors declare no competing financial interests.


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Author Contributions C.S.H. conceived the idea, carried out the experiments and data analysis, and wrote the manuscript. V.S. and O.V.K. designed the synthetic peptide library. G.B.M. contributed to writing of the manuscript.



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Figure 1 – The use of the IT detector for MS3 results in a significant reduction in proteome coverage. A 13-cell line mix digested with trypsin was labeled with TMT 6-plex reagents in a 1:1:1:1:1:1 pattern (TMT126 – TMT131) and spiked with a set of 550 synthetic peptides (3:3:3:1:1:1, TMT126 – TMT131). Samples were fractionated and analyzed on an Orbitrap Fusion using OT-MS3 or IT-MS3 detection. The scatter plot displays the relative quantification of the synthetic spike peptides (expected ratio 3:1) in the OT-MS3 and IT-MS3 samples. Dashed lines indicate the mean values of the spiked peptides in the noted data sets. Inset values display the number of spike peptides identified in both data sets, as well as the Pearson correlation between the quantification values. The table below displays metrics of the Human cell line peptide analyses with both detectors.

Table 1 – The numbers of MS2 spectra and subsequent peptide identifications can be improved using the IT for MS3 scanning. A 13-cell line mix digested with trypsin was labeled with TMT 6-plex reagents in a 1:1:1:1:1:1 pattern (TMT126 – TMT131) and spiked with a set of 550 synthetic peptides (3:3:3:1:1:1, TMT126 – TMT131). The values listed depict the mean of technical triplicate measurements (n = 3 injections). Values in parenthesis for MS2, Peptides, and Proteins indicate the change relative to the standard OT-MS2 sample (method A1).



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Table 2 – The numbers of MS2 scans can be increased using HCD fragmentation. A 13-cell line mix digested with trypsin was labeled with TMT 6plex reagents in a 1:1:1:1:1:1 pattern (TMT126 – TMT131) and spiked with a set of 550 synthetic peptides (3:3:3:1:1:1, TMT126 – TMT131). The values listed depict the mean of technical triplicate measurements (n = 3 injections). Values in parenthesis for MS2, Peptides, and Proteins indicate the change relative to the standard OT-MS3 sample (method A2). Mean Spike error is the average of the percent errors for the per peptide proportion of the reporter ion intensity assigned to the synthetic peptides in the specified samples.

Table 3 – The numbers of identifications and reporter ion yield are significantly impacted by the MS2 fragmentation energy. A 13-cell line mix digested with trypsin was labeled with TMT 6-plex reagents in a 1:1:1:1:1:1 pattern (TMT126 – TMT131) and spiked with a set of 550 synthetic peptides (3:3:3:1:1:1, TMT126 – TMT131). The values listed depict the mean of technical triplicate measurements (n = 3 injections). Values in parenthesis for MS2, Peptides, and Proteins indicate the change relative to the standard OT-MS3 sample with an NCE of 25 (method A8). Reporter signal is calculated as the mean of the intensity across all reporter ion channels across all peptides identified within a replicate.

Table 4 – The numbers of identifications and reporter ion yield are minimally impacted by the MS3 fragmentation energy. A 13-cell line mix


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digested with trypsin was labeled with TMT 6-plex reagents in a 1:1:1:1:1:1 pattern (TMT126 – TMT131) and spiked with a set of 550 synthetic peptides (3:3:3:1:1:1, TMT126 – TMT131). The values listed depict the mean of technical triplicate measurements (n = 3 injections). Values in parenthesis for MS2, Peptides, and Proteins indicate the change relative to the standard OT-MS3 sample with an NCE of 50 (method A14). Reporter signal is calculated as the mean of the intensity across all reporter ion channels across all peptides identified within a replicate.



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For TOC Only


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0

1

2

3

4

Relative Expression of Synthetic Spike Peptides in Analysis with OT and IT-MS3 Approaches

r2 = 0.42 n = 443

−1

log2(Fold Change 3:1 TMT Channels from IT-MS3)

Figure 1

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−1

0

1

2

3

log2(Fold Change 3:1 TMT Channels from

Sample Name

Method Index

OT-MS3 IT-MS3

Scan Setting Fragmentation

4 OT-MS3)

Detector

Mean and Relative Change Values

MS2

MS3

MS2

MS3

MS2

MS3

MS2

Peptides

Proteins

A2

Rapid

50K

CID

HCD

IT

OT

368,276

127,331

7,200

A3

Rapid

Normal

CID

HCD

IT

IT


204,251 (-80%) 61,678 (-106%) 5,900 (-22%)


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69x41mm (300 x 300 DPI)


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Investigating Acquisition Performance on the Orbitrap Fusion When

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