Electrospray ionization mass spectroscopic analysis of peptides modified with N-ethylmaleimide or iodoacetanilide Masoud Zabet-Moghaddam, Tomoko Kawamura, Emi Yatagai, Satomi Niwayama * Department of Chemistry and Biochemistry, Texas Tech University, PO Box 41061, Lubbock, TX 79409-1061, USA article info Article history: Received 1 June 2008 Revised 15 July 2008 Accepted 16 July 2008 Available online 23 July 2008 Keywords: Amino acid modifiers Peptides Proteomics Mass spectrometry abstract The cysteine-specific modifiers we reported previously, N-ethylmaleimide (NEM) and iodoacetanilide (IAA), have been applied to label cysteine residues of peptides in combination with electrospray ioniza- tion mass spectrometry (ESI-MS/MS), and their scope in proteomic studies was examined. Peptides mod- ified with N-ethylmaleimide (NEM) or iodoacetanilide (IAA) showed significant enhancement in ionization efficiencies. These modifiers were also found to remain intact in tandem mass spectrometry. Both combinations of N-ethylmaleimide (NEM) and d 5 -N-ethylmaleimide (d 5 -NEM), and iodoacetanilide (IAA) and 13 C 6 -iodoacetanilide ( 13 C 6 -IAA) were also shown to be applicable to quantitative analysis of a peptide. Ó 2008 Elsevier Ltd. All rights reserved. Proteomics, which studies sets of proteins expressed under cer- tain physiological conditions, is becoming important for solving problems in biology that involve the malfunction of complex pro- tein networks. In particular, identification and quantitative analy- sis of proteins found under different external stimuli are essential parts of proteomics research. Classical methods of quan- titative analysis include densitometric analysis 1 of 2D gels or radioisotope labeling. 2 More recent methods that have been pro- ven to be effective include application of stable-isotope labeling and subsequent mass spectrometric analysis for identification and concurrent quantitative analysis of proteins. 3 To this end, we have reported several sets of isotope-labeled and unlabeled small organic molecules that specifically react with cysteine residues. 4– 7 In combination with a soft ionization mass spectroscopy, MALDI TOF, we demonstrated that these sets of reagents allow quantita- tive analysis of proteins and peptides as well as identification of proteins, and therefore are expected to be a useful tool for proteo- mics research. While MALDI is a very common type of soft ionization mass spectrometry, ESI is also an equally common soft ionization meth- od for biochemical studies. While MALDI has certain advantages over ESI, such as ease of operation and tolerance to buffers and other additives, ESI also has certain advantages over MALDI, such as applicability to liquid samples, allowing direct connection to li- quid chromatography (LC), and non-interference from matrices. In addition, ESI is more commonly applied than MALDI to the studies of ligand–protein or protein–protein interaction. 8 Therefore, among the above cysteine-specific modifiers we pre- viously reported, we applied N-ethylmaleimide (NEM) and iodo- acetanilide (IAA) 9 to modification of model peptides and examined their effects by ESI-MS and ESI-MS/MS. In addition, we performed quantitative analysis of a model peptide using N-ethyl- maleimide (NEM) and d 5 -N-ethylmaleimide (d 5 -NEM), 1 and 2, as well as iodoacetanilide (IAA) and 13 C 6 -iodoacetanilide ( 13 C 6 -IAA), 3 and 4, in combination with ESI-MS in order to examine their applicability to proteomics studies. In particular, we compared the results with a well-known cysteine-specific modifier, iodoacet- amide (IA), 5, which has been commercially available for a long time. These modifiers, 1–5, are known to react with the sulfhydryl group of the cysteine residues as shown in Figure 1. Herein we re- port the results. First we examined the ionization efficiencies of model peptides with N-ethylmaleimide (NEM), 1, iodoacetanilide (IAA), 3, and iodoacetamide (IA), 5. Structural modifications of peptides can of- ten cause increase or decrease in the intensities of ionization effi- ciencies. 10 The enhancement of ionization efficiencies would allow detection of the peptides in smaller amounts, and therefore would be a great asset for proteomics research. With the use of ESI, we noticed significant reproducible enhancement by these modifiers. We therefore compared the enhancement with another well-known commercial cysteine modifier, iodoacetamide (IA). 10b,10c The three model peptides used were PEP 60, PEP 13, and PEP 31, of which the amino acid sequences, molecular weight, and pI val- ues are ALVCEQEAR, 1017.49 Da, 4.4; SDTCSSQKTEVSTVSSTQK, 2001.92 Da, 6.2; and KEEPPHHEVPESETC, 1746.75 Da, 4.5, respectively. Each peptide has one cysteine residue. The alkylation 0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2008.07.069 * Corresponding author. Tel.: +1 806 742 3118; fax: +1 806 742 1289. E-mail address: satomi.niwayama@ttu.edu (S. Niwayama). Bioorganic & Medicinal Chemistry Letters 18 (2008) 4891–4895 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl