Dataset Information

Removal of NHS-labelling by-products in proteomic samples

ABSTRACT: N-Hydroxysuccinimide (NHS) chemistry is used extensively across proteomics sample preparation. One of the most popular applications is its use in isobaric reagent based quantitation; specifically both the iTRAQ and TMT approaches. The labels are introduced onto primary Serines, tyrosines, and threonines can be labelled during TMT labelling reactions in addition to the desired amides. These peptides are typically classed as over-labelled and are disregarded for quantification. Their presence also unnecessarily increases sample complexity, which reduces identification rates. In this study, we developed a robust method to remove over-labelled peptides through systematic screening of nucleophilic aminolysis reagents and reaction conditions. The new method reduces the proportion of over-labelled peptides in the sample to less than 1% without affecting the labelling rate or introducing other modifications, leading to superior identification rates and quantitation precision.

INSTRUMENT(S): Orbitrap Ascend

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell, Cell Culture

SUBMITTER: Yana Demyanenko

LAB HEAD: Shabaz Mohammed

PROVIDER: PXD054730 | Pride | 2025-03-14

REPOSITORIES: Pride

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			Action	DRS
	2023-05-04-decoys-contam-UPR_Homo_sapiens_9606_220822.fasta.fas	Fasta
	Methylamie_optimisation_kinetic_sample_list.xlsx	Xlsx
	Methylamine_optimisation_kinetic_Fragpipe_search_results.zip	Other
	Methylamine_optimisation_kinetic_rawdata.zip	Other
	Optimised_workflow_validation_raw_data.zip	Other

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Project description:The investigation contains two sets of experiment. Set I. Transcriptional profiling of salt treated WT A. thaliana (WS ecotype) (100mM NaCl WT/0mM NaCl WT): Set II. Transcriptional profiling of salt treated ABR17- A. thaliana lines (100mM NaCl ABR17/0mM NaCl ABR17). Tissue for microarray analysis was obtained by placing surface sterilized seeds of A. thaliana (line 6.9) and the WT on half strength Murashige & Skoog (MS)(Murashige and Skoog, 1962) medium (1.5% sucrose, 0.8% agar with pH 5.7) medium in Petri dishes with or without 100mM NaCl at RT (21 ± 2 °C) after surface sterilization. The seeds were surface sterilized by rinsing with 70% ethanol for one minute, incubation in 20% bleach for 15 min and subsequent washes (four times for 5min each) to remove the bleach. MS plates with the seeds were placed at RT (21 ± 2 °C) under continuous fluorescent light 30 ?mol m-2 s-1 for 14 days. Seedlings (14 day-old) from three independently grown biological replicates in two set of experiments were removed from the MS plates, flash frozen in liquid nitrogen and stored at 80 °C until used for RNA extraction. Technical protocols for preparing the hybridization extract: 1. RNA was isolated using the QIAGEN RNeasy Plant Mini Kit (Qiagen Inc., Mississauga, ON, Canada) from two-week-old WT (grown on 0 and 100mMNaCl) and ABR17 (grown on 0 and 100mMNaCl) seedling tissue grown at three independent times (biological replicates). The integrity of all RNA samples assessed by agarose gel (1.2 percent) electrophoresis. 2. 6 ?g (Six micrograms) of total RNA was used to synthesize cDNAs using SuperScript® II RT (Invitrogen Inc., Burlington, ON, Canada) with RT polyA-capture primers in 3D Array 900TM (Genisphere Inc., Hatfield, PA, USA). Each pair of treated (100mM NaCl) and untreated (0mM NaCl) samples within each of the three biological replicates from two sets of experiments (100mM NaCl WT/0mM NaCl WT ; 100mM NaCl ABR17/0mM NaCl ABR17) was labelled in a reciprocal dye-swap design, for a total of 12 hybridizations. Note: In the transgenic plant production, the pea cDNA encoding for ABR17 was constitutively expressed under the control of Cauliflower mosaic virus 35S promoter. Reference: Srivastava S., Rahman H., Shah S. and Kav N.N.V. Constitutive expression of pea ABA-responsive 17 (ABR17) cDNA confers multiple stress tolerance in Arabidopsis thaliana. Plant Biotechnology Journal (2006) 4, 529-549.

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Removal of NHS-labelling by-products in proteomic samples

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