Neutralizing the Detrimental Effect of an N-Hydroxysuccinimide

Feb 6, 2018 - One of the most common chemistries used to label primary amines utilizes N-hydroxysuccinimide (NHS), which is also structurally incorpor...
1 downloads 0 Views 397KB Size
Subscriber access provided by MT ROYAL COLLEGE

Technical Note

Neutralizing the Detrimental Effect of an N-Hydroxysuccinimide Quenching Reagent on Phosphopeptide in Quantitative Proteomics Yumi Kwon, Shinyeong Ju, Prashant Kaushal, Jin-Won Lee, and Cheolju Lee Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04678 • Publication Date (Web): 06 Feb 2018 Downloaded from http://pubs.acs.org on February 7, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 22 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

Analytical Chemistry

Neutralizing the Detrimental Effect of an N-Hydroxysuccinimide Quenching

Reagent

on

Phosphopeptide

in

Quantitative

Proteomics Yumi Kwon,†,‡ Shinyeong Ju,†,‡ Prashant Kaushal†,§, Jin-Won Lee‡ and Cheolju Lee*,†, §



Center for Theragnosis, Korea Institute of Science and Technology, Seoul 02792, Korea



Department of Life Science and Research Institute for Natural Sciences, Hanyang University, Seoul

04763, Korea §

Division of Bio-Medical Science & Technology, KIST School, Korea University of Science and

Technology, Seoul 02792, Korea *

Corresponding author: Cheolju Lee

1

ACS Paragon Plus Environment

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

Page 2 of 22

Abstract One of the most common chemistries used to label primary amines utilizes Nhydroxysuccinimide (NHS), which is also structurally incorporated in various quantitative proteomic reagents such as isobaric tags for relative and absolute quantification (iTRAQ) and tandem mass tags (TMT). In this paper, we report detrimental effect of hydroxylamine, a widely used quenching reagent for excess NHS, on phosphopeptides. We found an impairment in the degree of phosphopeptide identification when hydroxylamine-quenched TMT-labeled samples were vacuum-dried and desalted compared to the non-dried (just diluted) and desalted ones prior to phosphoenrichment. We have also demonstrated that vacuum-drying in presence of hydroxylamine promotes β-elimination of phosphate groups from phosphoserine and phosphothreonine while having a minimalistic effect on phosphotyrosine. Additionally, we herein report that this negative impact of hydroxylamine could be minimized by direct desalting after appropriate dilution of quenched samples. We also found a 1.6fold increase in the number of phosphopeptide identifications after employing our optimized method. The above method was also successfully applied to human tumor tissues to quantify over 15,000 phosphopeptides from 3 mg of TMT 6-plex labeled-peptides. KEYWORDS:

hydroxylamine,

phosphoproteome,

phosphorylation,

hydroxysuccinimide-esters, tandem mass tag, isobaric tag

2

ACS Paragon Plus Environment

β-elimination,

N-

Page 3 of 22 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

Analytical Chemistry

Introduction Phosphorylation is a crucial component of signaling cascades in a cell and it plays a key role in various biological cellular functions such as cell growth, signal transduction, and metabolism. With rapid advances in high-resolution mass spectrometry (MS) instrumentation, new strategies and computational methodologies are on a constant uprise to study protein phosphorylation dynamics. As phosphorylation is a low stoichiometric post-translational modification, phosphopeptides (or phosphoproteins) need to be enriched from complex biological samples containing a large amount of non-phosphorylated background. In addition to the identification of phosphorylated proteins, quantitative phosphoproteomics is commonly employed to decipher the phosphorylation cascades in cell signaling pathways. Quantitative proteomic methods based on chemical labeling of stable isotopes employ mass tags such as TMT (tandem mass tags), iTRAQ (isobaric tag for relative and absolute quantitation) or ICPL (isotope-coded protein labels) to introduce quantitative information into peptides or proteins. In particular, the isobaric labeling tags such as iTRAQ and TMT have been widely used as a useful tool for bottom-up approaches1-4. Isobaric mass tagging enables simultaneous analysis of up to 8 (iTRAQ) or 11 (TMT) samples in one experiment and increases the starting amount of peptide by combining multiple samples prior to fractionation or phosphopeptide enrichment. It also minimizes the variation between samples occurred during the fractionation or phosphopeptide enrichment step, lowers MS1 complexity, and enables efficient use of instrument time. Like all other tags labeled on peptides, the isobaric tags are also labeled on the free amines of a peptide, e.g. α-amine at the N-terminus and/or εamine of lysine residue with the help of NHS-ester based chemistry. The NHS-ester is a highly aminereactive reagent and most widely used as bioconjugating reagent introduced nearly 40 years ago5,6. In practice, peptides of each sample are labeled with each different version of isobaric tags, and prior to combining labeled samples, quenching of excess tags are recommended in most of manufacturer’s instructions with the aim of preventing cross-contamination between the samples. 3

ACS Paragon Plus Environment

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

Although, there is no such recommendation in that of iTRAQ, quenching reagents routinely has been used after labeling step by various groups7-11. Simple chemicals containing primary amines, such as hydroxylamine, Tris, and glycine are generally used as a quenching reagent to deplete remaining NHS-ester reagent. Particularly, hydroxylamine also partially reverses over-labeling at unwanted positions, such as N-acylation of NHS at histidine or O-acylation at serine, threonine and tyrosine (mostly at tyrosine)

12,13

. Although the NHS-ester conjugation is most commonly used in quantitative

proteomics, no such study has been reported yet pinpointing the actual effect of isobaric labeling procedures on phosphopeptide identification. Hence in this study, we sought whether a chemical impact occurring during the TMT labeling reaction could be deleterious to phosphopeptide enrichment or not. In particular, we herein describe the impact of a reagent (hydroxylamine) intended for quenching excess TMT and suggest an optimized method towards increased phosphoproteome coverage in quantitative phosphoproteomics.

4

ACS Paragon Plus Environment

Page 4 of 22

Page 5 of 22 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

Analytical Chemistry

EXPERIMENTAL SECTION Labeling of TMT isobaric tags on peptides Tryptic peptides were prepared from HS683 cells and dried according to the procedure described in Supplemental Procedure. Dried peptides were re-suspended in 100 mM triethyl ammonium bicarbonate. Absorbance values at 280 nm were measured on a Nanodrop spectrophotometer (Thermo scientific) and the peptides were diluted to 1 mg/mL. An aliquot equivalent to 200 µg (200 µl) was taken and mixed with 1.6 mg of TMT0 label reagent (Thermo scientific) priorly prepared in 82 µl of acetonitrile (ACN) and incubated for 1 hr at room temperature. TMT labeling reaction was quenched by addition of 16 µl of 5% aqueous hydroxylamine.

Removal of excess TMT and quenching reagent a) Drying and desalting workflow Percentage of ACN in the aforementioned TMT labeling reaction mixture was about 29%. To remove excess amount of TMT reagent and hydroxylamine using solid-phase extraction, it was necessary to drop the hydrophobicity of sample solution beforehand. To do so, samples were dried in a vacuum centrifuge and then dissolved in 0.5% TFA and 5% ACN before application to HLB cartridge. b) Dilution and desalting workflow To decrease the ACN concentration, TMT reaction mixture was diluted with HPLC water to adjust ACN concentration to 5% and acidified to 0.5% TFA. The diluted sample was desalted on an HLB cartridge. Eluted TMT-labeled peptides were dried in a vacuum centrifuge to completion.

Phosphopeptide enrichment For all experiments, phosphopeptides were enriched with metal oxide affinity enrichment (MOAC) using titanium dioxide beads14,15 (10 µm, Titansphere Phos-TiO Bulk, GL Sciences, Tokyo, 5

ACS Paragon Plus Environment

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

Japan). The dried peptide (95% of starting amount) and TiO2 beads were pre-incubated separately in a solution of 3.45 M lactic acid (302 mg/ml), 60% ACN and 0.3 % TFA (200 µg peptide in 100 µL solution; 2 mg bead in 10 µL solution). The two pre-incubated mixtures were combined and further incubated for 30 min at 25°C with agitation. After incubation, beads with enriched phosphopeptides were collected by centrifugation and the supernatant was removed. The beads were washed with 1% TFA in 30% ACN and loaded onto a C8-plugged tip (Diatech Korea, Seoul, Korea). Bound phosphopeptides were eluted with 1.5 % NH4OH and then 5% pyrrolidine sequentially in a single tube. Eluates were directly acidified with 1% TFA and desalted using graphite spin columns (Thermo scientific) according to the manufacturer’s instructions.

Liquid chromatography and tandem mass spectrometry (LC-MS/MS) The phosphopeptide samples were reconstituted with 0.4% acetic acid and one fourth of each was analyzed with a reversed-phase C18 column (20 cm × 75 µm i.d., 3 µm, 120 Å, packed in-house; Dr. Maisch GmbH) on an Eksigent nanoLC-ultra 1D plus system. Before use, the column was equilibrated with 95% mobile phase A (0.1% formic acid in H2O) and 5% mobile phase B (0.1% formic acid in ACN). The peptides were eluted with a linear gradient from 5-40%B over 200 min followed by 80%B wash at a flow rate of 300 nL/min. The HPLC system was connected to a QExactive quadrupole mass spectrometer (Thermo Scientific, Bremen, Germany) operated in DDA mode. Survey full-scan MS spectra (m/z 300–1800) were acquired with a resolution of 70,000. Source ionization parameters were as follows: spray voltage, 2.5 kV; capillary temperature, 300°C; s-lens level, 44.0. The MS peak width at half height was