Peptide profiling of a single Locusta migratoria corpus cardiacum by nano-LC tandem mass spectrometry

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Peptides 24 (2003) 1475–1485

Peptide profiling of a single Locusta migratoria corpus cardiacum by nano-LC tandem mass spectrometry G. Baggerman a,∗ , E. Clynen a , J. Huybrechts a , P. Verleyen a , S. Clerens b , A. De Loof a , L. Schoofs a a

Laboratory for Developmental Physiology, Genomics and Proteomics, K.U. Leuven, Naamsestraat 59, 3000 Leuven, Belgium b Laboratory for Neuro-plasticity and Neuro-proteomics, K.U. Leuven, Naamsestraat 59, 3000 Leuven, Belgium Received 23 May 2003; accepted 19 August 2003

Abstract The pars intercerebralis–corpora cardiaca complex in insects is the functional equivalent of the vertebrate brain-pituitary axis. During the past few decades more than 40 neuropeptides have been isolated from the locust brain-corpus cardiacum complex. Tedious and time-consuming successive purification rounds of large tissue extracts were necessary to achieve the purification and sequencing of most of these signal molecules. Nowadays, the combination of nanoscale liquid chromatography and the very sensitive tandem mass spectrometry allows us to identify and sequence peptides in very low concentration directly from tissue extracts. In this manuscript, we review previous data on the peptidome analysis of the locust corpora cardiaca, with emphasis on AKH processing. In addition, we report the peptide profiling of a single corpus cardiacum from Locusta migratoria. 23 peptides were isolated and sequenced in a single nano-LC-MS/MS experiment, demonstrating the sensitivity and effectiveness of mass spectrometry in peptide research. © 2003 Elsevier Inc. All rights reserved. Keywords: Peptidomics; Insect; Q-TOF; Proteomics

1. Introduction During the past two decades, the knowledge on neuropeptides in insects has grown enormously. In locusts only, more than 40 neuropeptides have been characterized [54]. The majority of these peptides occur in the pars intercerebralis– corpora cardiaca complex. This complex is the functional equivalent in insects of the vertebrate brain-pituitary axis. Like the pituitary gland in vertebrates, the corpus cardiacum (CC) consists of a glandular lobe (CCg) and a neurohaemal or storage lobe (CCs). The glandular lobe (CCg) contains intrinsic neurosecretory cells that synthesize, store and release adipokinetic hormones (AKHs) and AKH precursorrelated peptides (APRPs) [20,21,35,52,56,58]. The neurohaemal part is the functional equivalent of the posterior lobe of the pituitary and is the storage and secretion site of several peptides produced in the neurosecretory cells of the brain as has been shown by immunocytochemical techniques [61]. These peptides include, FLRF-amide peptides, tachykinins, myotropins, crustacean cardioactive peptide, corazonin, ac-

Corresponding author. Fax: +32-16323902. E-mail address: [email protected] (G. Baggerman).

0196-9781/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2003.08.014

cessory gland myotropin, diuretic hormone, and myoinhibiting peptides. Many insect peptides, however, are structurally related and often cross immuno-reactivity impedes the identification of a particular peptide within a tissue of interest. New developments in mass spectrometry allow very sensitive and unequivocal identification of peptides present in extracts from individual organs or even in single cells or cell organelles [44]. In the past, a number of reports were published describing the direct analysis of a single organ with matrix-assisted laser desorption–time-of-flight mass spectrometry (MALDITOF MS) without prior preparation steps [13,29,32]. A similar MALDI-TOF method was used by Predel et al. [39], who performed a methanolic extraction to analyse the neuropeptide content of the abdominal perisympatic organs in cockroaches. Only in the recent few years, mass spectrometric techniques have been applied for de novo sequencing of neuropeptides from invertebrates using very limited amounts of starting material. Both electrospray quadrupole timeof-flight tandem mass spectrometry (ESI-Q-TOF MS/MS) [40] and MALDI-TOF MS post-source decay methods [26] proved to be successful. Both these techniques, however, have the disadvantage that abundant peptides present in the


G. Baggerman et al. / Peptides 24 (2003) 1475–1485

sample may reduce the signal intensity of the less abundant ones even to the point of making them undetectable. A solution to this problem is to fractionate the sample prior to its introduction into the mass spectrometer. Electrospray mass spectrometry is a very suitable ionization method to be combined with liquid chromatography, because the electrospray process transfers ions from solution directly into the gas phase. Coupling of an HPLC directly to the electrospray allows the analysis of the peptides as they elute from the column. After the electrospray technology was scaled down to the nanolevel, nanoscale capillary LC systems have been developed taking full advantage of low flow electrospray technology. Modern capillary HPLC systems can run gradients at flows as low as 100 nl/min to perform separations on columns with an internal diameter as small as 50 ␮m [55]. In a previous study, the combination of nano-LC and tandem mass spectrometry allowed us to identify 28 neuropeptides in an extract of 50 Drosophila melanogaster brains in a single analysis [2]. In locusts this approach is less straightforward to identify new peptides because in contrast to Drosophila no genome sequence is available from which identified peptides can be mined. Nevertheless, in a number of recent manuscripts, our group has reported the sequencing of new Locusta peptides using a similar LC-MS approach [3,9,10]. This manuscript reports on the use of capillary liquid chromatography–tandem mass spectrometry to identify neuropeptides from a limited amount of starting material, namely a single locust corpus cardiacum. 2. Material and methods 2.1. Animals and tissue preparation Locusta migratoria corpora cardiaca were dissected and extensively rinsed with NaCl solution (0.1 M). Extracts from one pair of corpora cardiaca were made by placing it in a microtube containing 10 ␮l of methanol/water/formic acid (90:9:1, v/v/v). The sample was gently shaken for 10 min; the remaining solid fraction was centrifuged down. The supernatant was removed and filtered with a spindown filter (Millipore). The filtrate was dried in a vacuum centrifuge and redissolved in 20 ␮l of acetonitrile/water/formic acid (2:97.9:0.1, v/v/v) prior to injection on the LC-MS system. Half of the total extract was injected on the LC-MS system.

C18, LC-Packings, The Netherlands). Ten microliters of the sample were loaded on the guard column with an isocratic flow of 2% acetonitrile in HPLC grade water, 0.1% formic acid, at a flow rate of 10 ␮l/min. After 2 min, the column-switching valve was switched, placing the guard column online with the analytical capillary column, a Pepmap C18, 3 ␮m 75 ␮m × 150 mm nanocolumn (LC Packings, The Netherlands). Separation was conducted using a linear gradient from 95% solvent A, 5% solvent B to 5% solvent A, 95% solvent B in 55 min (solvent A: water/acetonitrile/formic acid (94.9:5:0.1, v/v/v); solvent B: water/acetonitrile/formic acid (19.9:80:0.1, v/v/v)). The flow rate was set at 150 nl/min. The outlet of Ultimate capillary LC was connected to the electrospray interface of the Q-TOF mass spectrometer. The column eluent was directed through a metal-coated fused silica tip (Picotip type FS360-75-10 D, New Objective, USA). Needle voltage was set at 1250 V, cone voltage at 35 V. Tandem mass spectrometry was done in an automated fashion. The applied collision energy was chosen automatically depending on the number of charges and the mass to charge ratio of the selected ion.

3. Results 3.1. Nano-LC-MS peptide profiling The nano-LC separation resulted in a complex total ion current (TIC) chromatogram containing a high number of peaks (Fig. 1). The advantage of using a mass spectrometer as a detector for chromatography is that the total ion current (TIC) chromatogram is made from individual mass spectra. Most peaks in the chromatogram contain several compounds. However, modern tandem MS technology can select and fragment the peptide ions as they elute from the column, even when co-eluting with other peptides. The amino acid sequences of the selected peptides can then be deduced from the resulting fragmentation spectra. Only ions with a molecular mass of less than 3 kDa were selected for frag-

2.2. Capillary LC-tandem MS Capillary liquid chromatography–tandem mass spectrometry experiments were conducted using an Ultimate HPLC pump, a column-switching device (Switchos) and a Famos auto sampler (all LC Packings, The Netherlands) coupled to a Q-TOF hybrid quadrupole/time-of-flight mass spectrometer (Micromass, UK). Chromatography was performed using a guard column (␮-guard column MGU-30

Fig. 1. Total ion current (TIC) chromatogram obtained by nanoflow capillary LC tandem mass spectrometry from a single L. migratoria corpus cardiacum acidic methanolic extract. Neuropeptides identified by analysis of the CID fragmentation spectra or by mass solely are indicated.

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mentation since larger peptides cannot be fragmented in the collision cell of the Q-TOF. During the experiment more than 100 individual ions were automatically selected for fragmentation in the collision cell. Seventeen masses (Fig. 1; Table 1) could be attributed to known neuropeptides whereas other masses did not correspond to any of the locust peptide sequences known so far. 3.2. Adipokinetic hormones and their precusor-related peptides As could be expected from previous studies [8,36] the most abundant low mass peptide ions (mass
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