International Journal of Mass Spectrometry 376 (2015) 13–18 Contents lists available at ScienceDirect International Journal of Mass Spectrometry jou rn al h om epage: www.elsevier.com/locate/ijms Effect of laser intensity on the apparent isotope patterns of heme and peptide ions in MALDI-TOF MS Taehee Kim, Jihyeon Lee, Jeongkwon Kim ∗ Department of Chemistry, Chungnam National University, Daejeon, Republic of Korea a r t i c l e i n f o Article history: Received 16 September 2014 Received in revised form 23 October 2014 Accepted 2 November 2014 Available online 11 November 2014 Keywords: Mass spectrometry, Heme proteins, Heme b Heme c MALDI-TOF MS a b s t r a c t The apparent isotope patterns of heme ions and tryptically digested peptide ions in matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF) mass spectra (MS) were systematically investigated as a function of laser intensity. Two standard proteins, myoglobin and cytochrome c, were tryptically digested to provide heme b and heme c ions, respectively, along with several peptides. MALDI mass spectra of these materials, co-deposited with one of two matrix materials, 2,5-dihydroxybenzoic acid or -cyano-4-hydroxycinnamic acid (CHCA), were then generated using various laser intensities. For most peptides, the relative abundance of the second isotopic peak (SIP) to that of the monoisotopic peak was similar to the theoretical relative abundances. Regardless of laser intensity, the experimental SIP abundances of heme ions were consistently higher than the theoretical SIP abundances, which is due to the overlap of isobaric species. A gradual increase in the relative SIP abundance of the heme c ion was observed within a certain laser intensity range when the CHCA matrix was used with higher loadings (80 pmol) of tryptically digested cytochrome c, which is likely due to saturation of the MALDI-TOF MS detector. © 2014 Elsevier B.V. All rights reserved. 1. Introduction An iron-containing porphyrin is called a heme. Hemes are com- monly recognized as components of hemoglobin, myoglobin, and cytochrome c. Hemoglobin and myoglobin contain a noncova- lently bound heme group (heme b) while cytochrome c contains a covalently bound heme group (heme c) [1]. In mass spectrom- etry (MS), the heme b group is detected as [Fe(III) + porphyrin (C 34 H 32 N 4 O 4 Fe 1 )] + at m/z 616, the heme c group is detected as [Fe(II) + porphyrin + H (C 34 H 33 N 4 O 4 Fe 1 )] + at m/z 617 [1,2]. Our previous study investigated changes in the apparent isotopic distribution of heme ions as a function of the mass spectral acquisition mode [3]. The methods evaluated in that study were linear- and reflectron-mode matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF) mass spec- trometry (MS) and the low- and high-resolution modes of MALDI 15-T Fourier transform-ion cyclotron resonance (FT-ICR) MS [3]. The apparent isotope distributions of heme b and heme c ions changed as a function of acquisition mode due to the formation ∗ Corresponding author at: Department of Chemistry, Chungnam National University, 99 Daehak-Ro, Yuseong-Gu, Daejeon 305-764, Republic of Korea. Tel.: +82 428215477. E-mail address: jkkim48105@cnu.ac.kr (J. Kim). of metastable ions from the MALDI source and the overlap of isobaric species in low-resolution mode. For example, two additional ions, [C 34 H 31 N 4 O 4 Fe 1 ] + and [C 34 H 33 N 4 O 4 Fe 1 ] + , were observed in the MALDI 15-T FT-ICR MS spectrum of the heme b ion ([C 34 H 32 N 4 O 4 Fe 1 ] + ). Four such ions ([C 34 H 31 N 4 O 4 Fe 1 ] + , [C 34 H 32 N 4 O 4 Fe 1 ] + , [C 34 H 34 N 4 O 4 Fe 1 ] + , and [C 34 H 35 N 4 O 4 Fe 1 ] + ) were observed in the MALDI 15-T FT-ICR MS spectrum of the heme c ion ([C 34 H 33 N 4 O 4 Fe 1 ] + ). In a continuation of our previous investigation, this study describes the effect of laser intensity on the apparent isotopic distribution of heme ions and tryptically digested peptides from myoglobin and cytochrome c. The investigation on the effect of laser intensity on the isotopic distribution of heme ions is impor- tant since it can reveal whether the change of laser intensity can induce different forms of metastable ions or isobaric species. Myo- globin and cytochrome c are commonly used as standard proteins in method development [4,5]. To the best of our knowledge, this is the first report describing the influence of laser intensity on the isotopic distribution in MALDI. The investigation on the isotopic distribution in electrospray ionization (ESI) has been extensively performed [6–9]. The tryptic digests of myoglobin and cytochrome c were ana- lyzed by MALDI-TOF-MS at various laser intensities using either 2,5-dihydroxybenzoic acid (DHB) or -cyano-4-hydroxycinnamic acid (CHCA) as the MALDI matrix. http://dx.doi.org/10.1016/j.ijms.2014.11.005 1387-3806/© 2014 Elsevier B.V. All rights reserved.