Discrimination of Isoleucine and Leucine by Dimethylation-Assisted

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Discrimination of isoleucine and leucine by dimethylation-assisted MS3 Sheila Maibom-Thomsen, Søren Heissel, Ejvind Mørtz, Peter Hojrup, and Jakob Bunkenborg Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01375 • Publication Date (Web): 17 Jul 2018 Downloaded from http://pubs.acs.org on July 18, 2018

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Analytical Chemistry

Discrimination of isoleucine and leucine by dimethylation-assisted MS3

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Sheila Maibom-Thomsen1,2, Søren Heissel1, Ejvind Mørtz2, Peter Højrup1, Jakob Bunkenborg2.

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1: Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55,

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5230 Odense M, Denmark

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2: Alphalyse A/S, Unsbjergvej 4D, 5220 Odense SØ, Denmark

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*Corresponding author: [email protected]

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Abstract

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Protein sequencing by mass spectrometry has transformed the field of biopharmaceutical analysis but a

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missing part in the analytical toolkit is the ability to distinguish between the isomeric residues isoleucine

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and leucine since it is a requisite for efficient analysis of the primary structure of proteins. To address this

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need, we have developed a novel mass spectrometric method that combines reductive dimethylation and

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MS3 fragmentation with LCMS peptide mapping. The dimethylation of peptide N-termini leads to intense

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a1 ions upon collision induced fragmentation and further fragmentation of the isoleucine/leucine a1-ion

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leads to informative spectra with fragments that can discriminate between the two isomers. The

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methodology of a1-directed MS3 was applied to two antibodies in combination with the proteases trypsin,

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thermolysin, chymotrypsin and pepsin to generate peptides exposing N-terminal I/L residues.

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Introduction

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Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Determination of the complete amino acid sequence of proteins remains a challenge in analytical

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chemistry. Considerable effort is devoted to sequencing novel proteins with potential therapeutic benefit,

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such as new monoclonal antibodies, and for quality control monitoring of recombinant proteins. To

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establish the primary structure of proteins it is paramount to be able to distinguish between isoleucine and

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leucine residues. The classic method of Edman degradation1 utilizes the retention time difference of

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phenylthiohydantoin (PTH)-derivatized Ile/Leu residues. However, the two isomeric amino acids are not

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distinguishable by mass due to their identical elementary composition. Several mass spectrometry-based

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methods have been suggested to differentiate between Ile and Leu using dissociation methods that induce

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side chain fragmentation2–9. Fragmentation of the isomeric m/z 86 immonium ion from leucine and

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isoleucine yields a diagnostic ion at m/z 69 with higher intensity for isoleucine. For a given peptide

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sequence, this means that MSn can only be used to differentiate immonium ions from peptide fragments

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that contain a single Ile/Leu residue. Alternatively, ECD/ETD fragmentation of z-ions with

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leucine/isoleucine residues at the N-terminus can be used to distinguish between the isomeric residues.

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Here, leucine can fragment to a w-ion by isopropyl radical loss, whereas isoleucine predominantly loses an

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ethyl group thus leading to fragments with distinguishable mass. Bagal et al.7 developed a comprehensive

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LCMS method for de novo sequencing of monoclonal antibodies using a peptide sequence dependent

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decision tree combination of MS3 or side-chain losses of z-ions employing ETD-HCD that can be

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performed in a matter of days. Another approach combines germline homology data with the LCMS

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analysis after cleavage with chymotrypsin and leucine aminopeptidase that to some extent prefer leucine

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over isoleucine10.

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Chemical derivatization that preserves the primary structure of peptides is often used to encode reactive

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sites with the aim of extracting additional information. Dimethyl labeling adds two methyl groups to the α-

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amino group of N-terminal residues and the ε-amino group of lysine residues. The mass added by the


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methyl groups can be modified by using heavy stable isotopes, creating three compositionally different

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methyl groups; light (-CH3), intermediate (-CD2H) and heavy (-13CD3), which adds 28, 32 and 36 Da,

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respectively. Stable isotope dimethyl labeling is commonly used in quantitative proteomics, as it is fast,

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easy, and inexpensive to use and offers almost 100% complete labeling11. The intense a1 ion observed in

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HCD spectra of dimethyl labeled peptides reflects the N-terminal amino acid, and this information has

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proven valuable in peptide identification12, disulfide-bridge determination13 and PTM validation14. We

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noticed the similarity between the immonium ion from Ile/Leu residues and the dimethylated a1-ion. In

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this study, we measured the Ile/Leu a1-ion fragmentation pattern by MS3 of stable isotope dimethyl

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labelled reference peptides. The reference peptides were derivatized with three different isotopic dimethyl

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labelling forms to elucidate the fragmentation behavior of the I/L a1-ions. The MS3 fragment ion pattern

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of the dimethylated N-terminal I/L-residues showed fragmentation ions that can be used to discriminate

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between the isomeric side-chains. Based on these results, we created an LCMS method where a1-ion

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directed MS3 events are triggered by the presence of isomeric isoleucine and leucine a1 ions in the MS2.

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We tested the method on two antibodies and the analysis of the antibodies was performed with two

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different isotopic dimethyl labelling forms of four different proteolytic digests to test the method.

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Experimental

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Materials

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All chemicals were purchased from Sigma-Aldrich unless otherwise noted. The 6-peptide mixture was the

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Hi3 Phos B Standard from Waters (186006011). The monoclonal antibodies SiluTMLite SigmaMAb

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Universal human Antibody Standard was from Sigma Aldrich (Germany) and Trastuzumab was a gift from

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Chromacon (Switzerland)



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Methods

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Antibody digestion

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25µg of each monoclonal antibody was dissolved in Ultra-High Quality Milli-Q water (UHQ) (Elga,

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18.2MΩ), reduced in 10mM dithiothreitol (DTT) for 20min at 56°C and alkylated in 20mM iodoacetamide

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(IAA) for 30min at room temperature (RT) in the dark. For trypsin (Promega, USA) chymotrypsin

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(Worthington, USA) digestions, the antibody was diluted with UHQ and 1 M triethylammonium

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bicarbonate (TEAB) to a final protein concentration of 1µg/µL in 100mM TEAB. For thermolysin

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digestion the antibody was diluted with UHQ and 1M TEAB, 50mM CaCl2 (Merck Millipore, Germany) to

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a final protein concentration of 1µg/µL in 100mM TEAB, 5mM CaCl2. The sample used for pepsin

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digestion was acidified by diluting with 0.1% formic acid (FA) to a final protein concentration of 1µg/µL

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(pH approximately 3). 50µL immobilized pepsin (Thermo Fischer Scientific, USA) was added to the

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acidified sample. Digestion for the remaining samples were carried out with an enzyme to substrate ratio of

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1:20 (w/w). The trypsin, chymotrypsin and pepsin digestion samples were incubated at 37°C overnight. The

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thermolysin digestion sample was incubated at 70°C for 2h. Following digestion, the trypsin, chymotrypsin

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and thermolysin digested samples were split in 2 samples, each containing 12.5µg peptides, in preparation

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for dimethyl labeling. The liquid from the pepsin digestion sample was transferred to a new tube and the

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remaining resin washed with 1M TEAB, which was then added to the pepsin digestion liquid. The pH of

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this sample was checked and corrected with 1M TEAB until pH=8, and the sample was split in 2 for

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dimethyl labeling.

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Dimethyl labeling


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Hi3 PhosB peptides were dissolved in 25µL 100mM TEAB and 35pmol of each was reacted with the

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dimethyl labels. Light: 2µL 4% formaldehyde and 2µL 0.6M NaBH3CN was added. Intermediate: 2µL 4%

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formaldehyde-d2 (Isotec, USA) and 2µL 0.6M NaBH3CN was added. Heavy: 2µL 4% 13C-formaldehyde-d2

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and 2µL 0.6M NaBD3CN (Isotec, USA) was added. The samples were mixed and incubated at RT for 1h

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on a shaker. The labeling was quenched with 8µL 1% ammonia solution (EMD Millipore, Germany) and

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acidified for micropurification. The digested antibody peptides were diluted with 100mM TEAB to a final

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volume of 50µL. Light: 4µL 4% formaldehyde and 4µL 0.6M NaBH3CN was added. Intermediate: 4µL 4%

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formaldehyde-d2 and 2µL 0.6M NaBH3CN was added. The samples were incubated on a shaker at RT for

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1h. The labeling was quenched with 16µL 1% ammonia solution and acidified with FA for

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micropurification.

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Micropurification

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Reversed phase columns for micropurification were made in-house using Rainin GLS-L10 pipette tips

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(Mettler Toledo, Germany) containing Empore C18 lining (3M Company, USA) below 1.5-2cm Poros R2

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resin (20µm, Applied Biosystems, USA). The column was equilibrated with 100% acetonitrile (ACN),

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followed by 0.1% trifluoroacetic acid (TFA), the sample was loaded onto the column and washed with

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0.1% TFA before the peptides were eluted using 60% ACN, 0.1% TFA. All purified peptides were

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lyophilized.

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Mass Spectrometry

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Direct infusion MSn analysis of peptides

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The lyophilized peptide mixtures were dissolved in 50% ACN, 0.1% FA to a final concentration of

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100fmol/µL and transferred to a syringe and directly injected into a TSQ electrospray ionization (ESI)



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source (Thermo Scientific, Germany) coupled to an Orbitrap Fusion Lumos Tribrid mass spectrometer

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(Thermo Scientific, Germany), using a Chemyx Fusion 101 syringe pump (Thermo Scientific, Germany) at

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a flowrate of 3µL/min. MS1 was detected in the Orbitrap (resolution: 120.000, scan range: 400-1000m/z,

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RF lens: 30%, AGC target: 500.000, minimum injection time: 100ms, 1 microscan). Relevant peptide ions

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were manually selected for HCD MS2/MS3 analysis performed in the Orbitrap. For the unlabeled peptide

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mixture Ile/Leu immonium ions (m/z 86.09) were selected and HCD MS3 analysis was performed in the

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Orbitrap. For the dimethyl-labeled peptide mixtures, the a1 ions from the relevant peptides were selected

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for MS3 at m/z 114.13, 118.15 and 122.17 for the light, intermediate and heavy labeled samples,

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respectively, and MS3 events were performed on these in the scan range 50-150 m/z.

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nanoLC-MSn analysis of digested antibodies

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For antibody peptide analysis, the digested and dimethyl labeled peptides were dissolved in 0.1% FA

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(solvent A) and approximately 1µg peptides were analyzed using an EASY-nanoLC 1000 system (Thermo

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Scientific, Germany) coupled to the Orbitrap Fusion Lumos Tribrid mass spectrometer. The peptides were

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loaded onto a 2.5cm in-house packed Reprosil-Pur 120 C18-AQ (5µm: Dr. Maisch GmbH, Germany)

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precolumn with an internal diameter of 100µm and eluted directly onto a 19cm in-house packed Reprosil-

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Pur C18-AQ column (3 µm; Dr. Maisch GmbH, Germany) with an internal diameter of 75µm. A 104min

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HPLC gradient with a flowrate of 250 nL/min was used with a solvent B profile (95% ACN, 0.1% FA) in

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the following increments: 1-3% for 3min, 3-25% for 80min, 25-45% for 10min, 45-100% for 3min and

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100% for the final 8min. The peptides from each proteolytic digestion and each labelling form were run

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separately.

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The Orbitrap Fusion Lumos mass spectrometer was operated in data dependent acquisition mode with an

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MS1 Orbitrap scan (resolution: 120.000, scan range: 300-2000 m/z, AGC target: 400.000, maximum

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injection time: 100ms, RF lens: 30%, 1 microscan). For each 3s cycle, ions above a 50.000 intensity

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threshold and with a charge state of >+1 were selected for MS2. Monoisotopic peak determination was set

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to peptide and dynamic exclusion switched on (exclude after 1 time, exclusion duration: 15s, mass

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tolerance: ±10ppm, exclude isotopes). The selected MS2 precursors were isolated in the quadrupole at m/z

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1.0, fragmented using HCD at a collision energy of 30 and detected in the Orbitrap (resolution: 30.000,

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first mass: 100 m/z, AGC target: 200.000, maximum injection time: 54s). For each MS2 event one m/z

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targeted trigger is assigned; 114.1283 m/z for the light and 118.1526 m/z for the intermediate dimethyl

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labeled peptides. The presence of the targeted fragment triggers an MS3 event of the ion with scan range

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50-125 m/z. The MS3 precursor is fragmented by HCD and analyzed by the Orbitrap (MS1 and MS2

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isolation window: 0.8 m/z, Collision energy: 30%, AGC target: 5.0e4, Resolution: 15.000, maximum

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injection time: 200ms, first mass: 50 m/z, 1 microscan).

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Data analysis.

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MS raw files were processed with Proteome Discoverer (version 2.0). The MS2 peak lists were searched

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using Mascot (v. 2.4.1) against a FASTA database containing the SiluMab and Trastuzumab antibody

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protein sequences and the protease sequences (10 entries). Carbamidomethylation of cysteines and

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light/medium dimethylation of N-termini and lysine residues were set as fixed modifications and oxidation

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of methionine and deamidation of asparagine as variable modifications. Enzyme specificity was set as

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None and peptide identification was performed with a precursor mass tolerance of 10 p.p.m. and fragment

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mass tolerance of 0.05 Th. The MS3 spectra were extracted with a python script using Multiplierz14 and the

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intensities from the MS3 peak lists were used to assign fragment ion intensities: IIle (light) = Imz 69 + Imz 85 +



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Imz 99 ; IIle (medium) = Imz 69 + Imz 89 + Imz 103; ILeu(light) = Imz 72 ; ILeu(medium) = Imz 76. The I-ratio is calculated

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for peptides with IIle + ILeu > 0 as IIle /( IIle + ILeu). The I/L score was calculated for peptides with a Mascot

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score above the Identity threshold and in most cases each assignment was supported by multiple

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observations of the I/L- residue from different peptide sequences, modification and charge states. The

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mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the

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PRIDE15 partner repository with the dataset identifier PXD009295 and 10.6019/PXD009295

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Results and discussion

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To examine the fragmentation pattern, we performed three different states of stable isotopic dimethyl

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labeling on a standard peptide mixture (Hi3 PhosB, Waters). The derivatized peptides were analyzed by

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direct infusion on an Orbitrap Fusion Lumos, which has a lower mass range limit at m/z 50. MS3 spectra

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of the a1 ions from the dimethyl labeled peptides IGEEYISDLDQLRK and

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LITAIGDVVNHDPVVGDR are shown in Figure 1 a and b, respectively. Both Leu and Ile MS3 spectra

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share a common fragment at m/z 58.065, 62.091 and 66.110 for the light, intermediate and heavy labeled

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peptides, respectively. The fragmented isoleucine a1 ion has two intense unique fragments: a peak at m/z

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69.070 that is constant in all labeling conditions and a peak from the loss of 29 Da from an ethyl group. In

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addition, a less intense fragment originates from the loss of 15 Da from a methyl group. The leucine a1

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MS3 HCD spectrum shows an intense diagnostic peak corresponding to the loss of a 42 Da propylene

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group rather than the loss an isopropyl group. The proposed bond cleavages are shown in Fig. 1c and the

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corresponding fragment masses are tabulated in Fig. 1d.

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We chose two monoclonal antibodies (mAbs) with known sequence to test the methodology. SILuLite and

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Trastuzumab were digested, using the proteases trypsin, chymotrypsin, thermolysin, and pepsin,

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respectively, to generate peptides with N-terminally exposed I/L. The digested peptides were dimethyl-

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labeled with formaldehyde(light) and deuterated formaldehyde (intermediate). Each proteolytic digest and

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labelling state was analyzed separately by nanoLC-MS and to limit the number of samples we did not

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include the heavy label for this proof-of-concept analysis. Only one labelling state is necessary for the I/L

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discrimination experiment but the experimental setup with two separate isotopic modifications of the

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amine groups of the N-terminus and lysine residues has the advantages that the modifications can be

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considered as fixed modifications due to the efficient chemistry of the dimethyl labelling and that the

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parent and fragment ion assignments can be queried for consistency. We included a branch in the

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acquisition scheme (Figure 2c), so that if an HCD tandem MS spectrum contained the diagnostic a1 ion

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from I/L, this ion would be selected for HCD MS3 fragmentation. An example is shown in Figure 2 a,b

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where the MS2 data of the dimethylated peptide can be confidently assigned and the intense a1-ion triggers

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an MS3 event. Intensities from the MS3 peak lists were used to create a simple metric: IIle (light) = Imz 69 +

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Imz 85 + Imz 99 and ILeu(light) = Imz 72. The masses are shifted according to the stable isotope labeling scheme

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and the I-ratio is calculated as IIle /( IIle + ILeu). Spectra with I-ratio greater than 0.8 are assigned as

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isoleucine and with a ratio less than 0.2 as leucine. The MS3 spectrum in Fig 2b has an I-ratio of 0 and is

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assigned as leucine in agreement with the peptide identification. Spectra with an I-ratio between 0.2-0.8

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with mixed intensities from I and L a1-ions are not used for classification to avoid cases where there is a

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contribution from an a1-ion from co-eluting peptides within the MS2 precursor selection m/z window.

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Running two different states of dimethyl labelling changes the MS2 selection window and reduces the

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likelihood of having consistently confounding co-eluting peptides in both states. Using these criteria, we

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did not observe any inconsistent assignment of I/L between the two labelling forms. The peptide-to-



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spectrum matches with an N-terminal I or L residue identified by Mascot database searching are

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documented in Supplementary Table 1 along with the a1-directed MS3 spectrum associated to it. Using this

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approach and the limited set of proteases, we could assign and confirm the identity of between 71-94% of

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the isoleucine/leucine residues in each subunit (Figure 2d).

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A significant advantage of the dimethylation prior to ionization and fragmentation is that it ensures that

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short peptides do not cyclize and scramble16 which can confound multistage CID-based analysis. Likewise,

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by fixing the readout to the N-terminal amino acid, the method does not suffer from the radical site

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migration problem, that has been reported for w-ion based isoleucine/leucine discrimination9. The radical

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site migration appears when applying both ETD and collisional activation and can be reduced by

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minimizing the applied normalized collision energy9. A limitation of the proposed method is that only

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Ile/Leu residues exposed at peptide N-termini can be distinguished. Finding means of exposing all leucine

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and isoleucine residues in a protein as N-terminal residues on peptides well suited for LCMS analysis will

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require optimization of digestion conditions and enzymes. Possibly this limitation can be circumvented

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because labeling the N-terminal residue by dimethylation changes the immonium ion of the first residue

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and the remaining residues could be analyzed in combination with a m/z 86 immonium ion based methods

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for peptides that contain both an N-terminal and an internal Ile/Leu residue.

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The presented method to distinguish Leu and Ile residues in peptide LC MS analysis can be extended by

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increasing the panel of proteases and digestion conditions. To reduce analysis time it is possible to pool

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different proteases digestions prior to analysis with the caveat that increasing complexity will increase the

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likelihood of mixed I/L spectra from co-eluting co-fragmenting peptides. While not necessary for the I/L-


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discrimination the use of stable isotope labelling allows the peptide identifications and ion assignments to

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be experimentally tested by the observation of mass changes consistent with the proposed peptide

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sequence. The sensitivity of the method benefits from the altered fragmentation pattern caused by the

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dimethylation which increases the signal of the a1 ion and the simplicity of the resulting data lends itself to

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automation. Since the discrimination of Ile/Leu is independent of peptide identification and does not need

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sequence dependent fragmentation, the method can easily be applied to de-novo sequencing of proteins.

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Figure 1.



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MS3 fragmentation of dimethylated Ile/Leu a1-ion. (a,b) The a1 ion MS3 HCD-fragmentation spectra

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of the dimethyl labeled peptides IGEEYISDLDQLRK (a) and LITAIGDVVNHDPVVGDR (b) using

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three different stable isotope dimethylation labels. (c) The bonds proposed cleaved to produce the

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observed fragment ions. (d) Theoretical mass values for each label state. The dimethylation sets an N-

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terminal Ile/Leu residue apart from internal Ile/Leu immonium ions and the added mass of the stable

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isotopic dimethyl labeling increases the set of differentially diagnostic ions to mass values above the

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instrument cut-off at m/z 50 during MS3 fragmentation of the a1-ion.

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Figure 2

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Discriminating Ile/Leu during LCMS analysis. (a) HCD MS/MS spectrum of the dimethyl-labeled

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peptide identified as LFPPSSEELQANK. (b) The MS3 spectrum triggered by the presence of the intense


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a1 ion. The presence of the m/z 72 ion - and the absence of isoleucine-specific fragment ions – identifies

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the N-terminal residue as leucine. (c) Acquisition scheme for the LCMS analysis. (d) Summary of the

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combined identifications from all proteases and labeling states.

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Supporting information available:

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Table of MS3 I/L identifications

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Data are available via ProteomeXchange with identifier PXD009295.

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Acknowledgements

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The authors thank the Innovation Fund Denmark (5016-00002B) for the financial support, and the Villum

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Center for Bioanalytical Sciences. We thank ChromaCon AG for providing the Antibody Trastuzumab.

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Competing financial interests

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The authors declare no competing financial interests.

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References

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(1) (2)

Edman, P. Method for Determination of the Amino Acid Sequence in Peptides. Acta Chem. Scand. 1950, 4, 283–293. Johnson, R. S.; Martin, S. A.; Biemann, K.; Stults, J. T.; Watson, J. T. Novel Fragmentation Process of Peptides by Collision-Induced Decomposition in a Tandem Mass Spectrometer: Differentiation of Leucine and Isoleucine. Anal. Chem. 1987, 59 (21), 2621–2625.



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(5) (6) (7) (8) (9) (10)

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Table of Contents Figure


Discrimination of Isoleucine and Leucine by Dimethylation-Assisted

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