A longitudinal proteomic assessment of peptide degradation and loss under acidic storage conditionsAnalytical Biochemistry

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Authors
Melanie Planyavsky, Marie L. Huber, Nico A. Staller, André C. Müller, Keiryn L. Bennett
Year
2015
DOI
10.1016/j.ab.2014.11.020
Subject
Molecular Biology / Biochemistry / Biophysics / Cell Biology

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Peptide storage o an volv ons rage des at t nd, ge of eral m s, Aus simila comp aining nd Nffers a shelf peptides but are difficult to apply to the mixture of peptides mation of a cyclic imide intermediate can lead to cleavage of the acid followed by clic interm

To a lesser orm cyclic

Deamidation occurs in peptides containing the sequence asparagine or glutamine followed by a glycine residue. The deamidation of the asparagine–glycine sequence results in the formation of a cyclic imide intermediate that is hydrolyzed to form the aspartate or isoaspartate analogue of asparagines. In addition, the cyclic imide intermediate can lead to racemization into D-aspartic acid or D-isoaspartic acid analogues of asparagine. Cysteine and methionine residues are the predominant residues that undergo ⇑ Corresponding author. Fax: +43 1 40160 970000.

E-mail address: kbennett@cemm.oeaw.ac.at (K.L. Bennett). 1 These authors contributed equally to the work. 2 Abbreviations used: LC, liquid chromatography; ESI, electrospray ionization; MS, mass spectrometry; BSA, bovine serum albumin; 6BM, bovine 6 protein tryptic digest equimolar mixture; RT, room temperature; HPLC, high-performance liquid chromatography; SU, single use; FT, freeze–thaw; CID, collision-induced dissociation; IQR, interquartile range.

Analytical Biochemistry 473 (2015) 11–13

Contents lists availab

Analytical Bio journal homepage: www.e20 or 80 C over a range of time periods. Reasons for different intermediates that eventually also result in cleavage of the peptide.generated for a proteomic experiment. Prior to analysis by liquid chromatography electrospray ionization mass spectrometry (LC–

ESI–MS)2, peptide samples are prepared according to widely accepted strategies and are often stored under acidic conditions at peptide chain. Similarly, the presence of aspartic a glycine residue can result in hydrolysis of the cy to aspartic acid or into an isoaspartate analogue. peptides containing serine residues can also fhttp://dx.doi.org/10.1016/j.ab.2014.11.020 0003-2697/ 2014 Elsevier Inc. All rights reserved.ediate extent, imideand freezing aliquots will extend the storage life of the peptide, and storage at 20 C or lower is optimal. Repeated freeze–thaw cycles should be avoided because this can degrade the peptides.

Any remnants of a thawed aliquot should be discarded.

Naturally, these guidelines are designed for synthetic off-the[1], (ii) deamidation [2,3], (iii) oxidation [4], and (iv) 2,5-diketopiperazine and pyroglutamic acid formation [5]. Hydrolysis can occur in peptides containing aspartic acid, whereby the amino acid can dehydrate to form a cyclic intermediate. If a peptide contains an aspartic acid residue followed by proline, the acid-catalyzed for-Storage temperature

Orbitrap Velos mass spectrometer

Proteomics

The recommendations for stora synthetic peptides in solution by sev

Sigma–Aldrich, GenScript, Mimotope

Peptides, and Activotec, are all very of peptides in solution is limited samples. In particular, peptides cont cysteine, methionine, tryptophan, a are unstable. The use of sterile bucommercially available anufacturers, including

Pep, Proimmune, Think r. Namely, the shelf life ared with lyophilized asparagine, glutamine, terminal glutamic acid t neutral pH (pH 5–6) storage conditions may be due to the availability of machine analysis time or surplus samples stored after analysis for reassessment at a later time point if required. Questions arose in our laboratory regarding the stability of tryptic peptides generated for proteomic experiments in acid under medium- to long-term storage.

The stability of a peptide in solution can vary depending on the amino acid composition. Some of the possible routes by which peptides can be chemically degraded are through (i) hydrolysisPeptide stability

Acid degradation  2014 Elsevier Inc. All rights reserved.Notes & Tips

A longitudinal proteomic assessment of p under acidic storage conditions

Melanie Planyavsky 1, Marie L. Huber 1, Nico A. Stall

CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, 10 a r t i c l e i n f o

Article history:

Received 25 August 2014

Received in revised form 19 November 2014

Accepted 20 November 2014

Available online 3 December 2014

Keywords: a b s t r a c t

Sample preparation prior t try (LC–ESI–MS) usually in iable time periods. Questi medium- to long-term sto on storage of tryptic pepti from this evaluation is th experiments is at 80 C aptide degradation and loss 1, André C. Müller, Keiryn L. Bennett ⇑ ienna, Austria alysis by liquid chromatography electrospray ionization mass spectromees the storage of frozen peptide samples in an acidic environment for vararose in our laboratory regarding the stability of peptides in acid under . Thus, a 10-month longitudinal study was designed to assess the effect at 20 and 80 C under acidic conditions. Our conclusion and proposal he optimal storage conditions of peptide samples in acid for proteomic ideally, as separate aliquots. le at ScienceDirect chemistry lsevier .com/locate /yabio

The MS analyses were performed in a data-dependent acquisition mode using a top 15 collision-induced dissociation (CID) method. Dynamic exclusion for selected ions was 60 s. The maximal ion accumulation times for MS in the Orbitrap and MS2 in the LTQ were 500 and 50 ms, respectively. In MS and MS2 modes, automatic gain control (AGC) was set to 106 and 5000 ions, respectively. Peptides were detected in MS mode at a resolution of 60,000 (at m/z 400). All samples were analyzed every 4 weeks for a period of 10 months as technical back-to-back triplicates.

The acquired MS data files were processed with Skyline 2.5 (version 2.5.0.6157) [7]. A reference .raw MS file from each data set was converted to a .dat file with Mascot (version 2.3.2) to build a suitable library for Skyline. The generated library was matched with a FASTA file containing sequence data of the proteins in the sample. Mass tolerances on the precursor and fragment ions were ± 0.5 m/z. Two missed tryptic cleavage sites were allowed. Carbamidomethyl cysteine (BSA) and carboxymethyl cysteine (6BM) were fixed modifications. Oxidized methionine and tryptophan, deamidated glutamine and asparagine, N-terminal pyroglutamic acid, and N-terminal deaminated pyroglutamyl were variable modifications.