Peptide sequencing using chemically assisted fragmentation (CAF) and Ettan MALDI-ToF Pro mass spectrometry
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1 application note Ett MALDI-ToF Pro Peptide sequencing using chemically assisted fragmentation (CAF) d Ett MALDI-ToF Pro mass spectrometry Key words: MALDI-ToF, PSD, peptide sequencing, chemically assisted fragmentation Introduction Peptide mass fingerprinting using matrix-assisted laser desorption/ionization (MALDI) time-of-flight (ToF) mass spectrometry has become a major tool for identifying proteins in proteomics research. The method provides high speed, sensitivity, d mass accuracy, but for a substtial fraction of the proteins alyzed, identification is not definitive. Under these circumstces, amino acid sequence information from one or more peptides is required for unambiguous identification. Such information c be readily obtained using the Ett TM CAF-MALDI Sequencing Kit in conjunction with Ett MALDI-ToF Pro mass spectrometer (Fig 1). Ett MALDI-ToF Pro has a quadratic field reflectron that allows fast post-source decay (PSD) alysis, focusing all fragments in a single run, independent of size. The accompying software includes tools for automated protein identification from data generated using the Ett CAF-MALDI Sequencing Kit. The chemistry of the Ett CAF-MALDI Sequencing Kit, based on a new class of water-stable sulfonation reagents, eliminates the most common problems associated with PSD alysis. Ett CAF-MALDI chemistry greatly improves the fragmentation efficiency of the peptides d also simplifies the interpretation of their fragmentation spectra. The special features of the Ett MALDI-ToF Pro mass spectrometer d the simple d robust derivatization protocols using the Ett CAF-MALDI Sequencing Kit combine to provide the proteomics researcher with: Identification of increased numbers of proteins Rapid, sensitive, d precise peptide sequencing Increased number of proteins identified by MALDI-ToF Ett CAF-MALDI Sequencing Kit Peptide sequencing Characterization of phosphorylation sites Fig 1. Ett CAF MALDI Sequencing Kit in conjunction with Ett MALDI-ToF Pro offers a multifaceted approach to protein identification. Strategy CAF-MALDI chemistry is based on the introduction of a negatively charged group to the N-terminus of peptides generated by tryptic digestion. The theory is that, after derivatization, the formation of a positively charged ion (net charge) requires that two protons be introduced into the peptide. ne of these protons will primarily reside in the basic C-terminal side chain, while the other has a higher degree of freedom to resonate in the peptide backbone, assisting fragmentation. In the appropriate alysis mode, only the y-ions, which retain a net positive charge, will become separated in the reflectron. The N-terminal fragment ions will be neutral d non-detectable. The derivatization reaction is divided into two steps (Fig 2). The first step converts the e-amino group of each lysine side chain to homo-arginine (generating a mass addition of 42 atomic mass units [amu]). This step is necessary to protect the lysine side chains from sulfonation in the following step. The second step introduces a sulfonic acid group at the N- terminus (generating a mass addition of 136 amu). Characterization of phosphorylation sites AA, p1
2 Ett MALDI-ToF Pro N NH CH C NH 2 lysine H 2 N CH 3 Fig 2. The two steps of the derivatization reaction. Products used Code no. Ett CAF-MALDI Sequencing Kit Kit contents: Lysine modifier: -methylisoureahydrogen sulfate CAF reagent: NHS-ester containing a sulfonic acid group Stop solution: 50% hydroxylamine solution Lysine modifier buffer: 0.25 M sodium carbonate, ph 11.7 CAF buffer: 0.25 M sodium bicarbonate, ph 9.4 Control peptides: Lys-peptide: HGTVVLTALGGILK (M r 1378) Arg-peptide: ADSGEGDFLAEGGGVR (M r 1536) ACH-Cinnamic Acid, Ett Chemicals Trifluoroacetic Acid, Ett Chemicals Methods d Instrumentation Derivatization reactions of tryptic peptides were performed using the instructions d reagents in the Ett CAF-MALDI Sequencing Kit. Additional reagents included C 18 ZipTip (Millipore), ACH-Cinnamic Acid, Trifluoroacetic Acid, acetonitrile (Sigma), d ultrapure water (18 MW/cm). Samples d matrix were loaded on the Ett MALDI-ToF Pro target as described in the kit instructions. All spectra were obtained with the ion gate set for the precursor ion (i.e. parent ion) in PSD alysis mode. C NH NH CH C The fragment masses of CAF-labelled peptides were determined using the Ett MALDI-ToF Pro software package. Proteins were identified using automated protein identification program included in the system. Several types of alysis were performed to show examples of the performce of the Ett CAF-MALDI Sequencing Kit in peptide sequencing d identification of phosphorylation sites, as well as to provide data on sensitivity with different samples. H 2 N NH homoarginine (+42 amu) Step 1. Protection (guidination) of the ε-amino group of lysine. R 1 S H + H 2 N -methylisoureahydrogen sulfate H Pep Step 2. Derivatization of the N-terminus using the CAF reagent. R 2 C NH H S H R 1 NH Pep R 2 sulfonated N-terminus (+136 amu) Results d Discussion Peptide sequencing The simplified PSD spectra, showing y-ions only, allow fast d accurate sequence determination, where the distce between two consecutive peaks corresponds to the mass of amino acid. Figure 3 shows example of the complete peptide sequencing of a model peptide comprising 16 amino acids. 2,0 2,0 2,000 1,800 1,0 1,0 1,0 1, R V G G G E A I/L F D G E G S D A ,000 1,0 1,0 1,0 Fig 3. Peptide sequencing of a model peptide comprising 16 amino acids. Fig 4. Peptide sequencing of a peptide containing two phosphorylated tyrosine residues. The sequence is ALGADSpYpYTAR (two fragment peaks are missing from the spectrum, each indicated by X) CAF label ,800 Identification of phosphorylation sites The Ett CAF-MALDI Sequencing Kit c also be used for rapid identification of phosphorylation sites. An example of peptide sequencing of a phosphorylated peptide is shown in Figure 4. The peptide contains two phosphorylated tyrosine residues. The phosphorylated tyrosine residues remain intact during PSD d c easily be identified by a mass difference of 243 amu ( , HP 3 ) between them R X X py py S D A G I/ A = = AA, p2
3 Ett MALDI-ToF Pro Sensitivity The sensitivity limit, defined as the lowest amount of sample needed for protein identification from CAF-MALDI data, was determined for both lysine- d arginine-terminated peptides from different sources. Model peptides, in-solution, d in-gel digests were all investigated. Figure 5 shows the results when fmol of a lysine-terminated peptide was alyzed. Figure 6 shows the results with fmol of arginine-terminated peptide. In both cases, the sequence could be determined d the protein identified. The sensitivity limit for in-solution digest of horse myoglobin was fmol on the MALDI target for both lysine- d arginine-terminated peptides (see Figs 7A d B) fmol of a lysine-terminated model peptide 35 fmol of arginine-terminated model peptide A :0:9 Fig 5. Sensitivity of a Lys-terminated model peptide (containing the same sequence as a Lys-terminated tryptic peptide of horse myoglobin). Four hundred fmol were derivatized, d one-tenth of the sample ( fmol) was alyzed. Fig 6. Sensitivity of Arg-terminated model peptide (containing the same sequence as Arg-terminated tryptic peptide of fibrinopeptide A). Four hundred fmol were derivatized, d onetenth of the sample ( fmol) was alyzed AA, p3
4 Ett MALDI-ToF Pro Finally, phosphorylase B was used to investigate the sensitivity limit for in-gel digest. Seven-hundred d fifty fmol of protein was spiked into a gel plug. The protein was digested with trypsin d the peptides were thereafter extracted from the gel plug, followed by lysine protection d CAF-derivatization. Figure 8 shows the PSD spectrum of one of the peptides of the digest, when one tenth of the sample was loaded onto the MALDI target. Fig 7a. Fig 7b. fmol of arginine-terminated tryptic peptide from in-solution digest fmol of a lysine-terminated tryptic peptide from in-solution digest ,000 1,0 1,0 1,0 1,0 1,500 1,0 Fig 7a-b. Analysis of fmol of a trypic peptide of horse myoglobin from in-solution digest. In Pel A, the peptide is Arg-terminated, d in Pel B it is Lys-terminated. In both cases, 0 fmol were derivatized, d one-tenth ( fmol) was alyzed AA, p4
5 Ett MALDI-ToF Pro fmol of arginine-terminated tryptic peptide from a gel Conclusions Ett CAF-MALDI Sequencing Kit in combination with single-run PSD alysis using Ett MALDI-ToF Pro d its automated software for protein identification from fragmentation data enables sensitive d rapid peptide sequencing of tryptic peptides. Key advtages of the kit include: Specific d complete N-terminal derivatization under aqueous conditions. ptimized derivatization protocol for fast d robust sample preparation. Increased fragmentation in PSD PSD spectra allow easier interpretation of data. Precise identification of phosphorylation sites. High sensitivity. Low fmol levels of model peptides, in solution or in-gel digests c be used for protein identification by PSD using the Ett CAF-MALDI Sequencing Kit. References 1. Keough, T. et al., A method for high-sensitivity peptide sequencing using post-source decay matrix-assisted laser desorption ionization mass spectrometry. Proc. Natl. Acad. Sci. USA. 96, (1999). 2. Liminga, M. et al., Water-stable chemistry for improved amino acid sequencing by derivatization post-source decay (dpsd) using Ett MALDI-ToF with a quadratic field reflectron. Proc. 49 th Conf. Mass Spectrometry d Allied Topics, Chicago, IL (01). 3. Keough, T. et al., Atmospheric pressure matrix-assisted laser desorption/ionization ion trap mass spectrometry of sulfonic acid derivatized tryptic peptides. Rapid Commun. Mass Spectrom. 15, (01). Fig 8. Sequencing of Arg-terminated tryptic peptide of phosphorylase B isolated from a 2-D gel. Seven-hundred d fifty fmol were derivatized, d one-tenth of the protein from the gel was alyzed AA, p5
6 Ett MALDI-ToF Pro to order: Asia Pacific Tel: Fax: Australasia Tel: Fax: Austria Tel: Fax: Belgium Tel: Fax: Cada Tel: Fax: Central, East, South East Europe Tel: Fax: Denmark Tel: Fax: Finld & Baltics Tel: +358 (0) Fax: +358 (0) Frce Tel: Fax: Germy Tel: Fax: Italy Tel: Fax: Jap Tel: Fax: Latin America Tel: Fax: Middle East d Africa Tel: + (1) Fax: + (1) Netherlds Tel: Fax: Norway Tel: Fax: Portugal Tel: Fax: Russia & other C.I.S. & N.I.S. Tel: +7 (095) , Fax: +7 (095) South East Asia Tel: Fax: Spain Tel: Fax: Sweden Tel: Fax: Switzerld Tel: Fax: UK Tel: Fax: USA Tel: Fax: Ett is a trademark of Amersham Biosciences Limited. Amersham d Amersham Biosciences are trademarks of Amersham plc. ZipTip is a trademark of Millipore Corporation. Amersham Biosciences AB Björkgat, SE Uppsala, Sweden. Amersham Biosciences UK Limited Amersham Place, Little Chalfont, Buckinghamshire HP7 9NA, Engld. Amersham Biosciences Corporation 800 Centennial Avenue, P Box 1327, Piscataway, NJ USA. Amersham Biosciences Europe GmbH Munzinger Strasse 9, D Freiburg, Germy. Amersham Biosciences KK Sken Building, , Hyakunincho, Shinjuku-ku, Tokyo , Jap. All goods d services are sold subject to the terms d conditions of sale of the compy within the Amersham Biosciences group that supplies them. A copy of these terms d conditions is available on request. Amersham Biosciences AB 02 All rights reserved. Produced by Wikströms, Sweden 379, Printed matter. Licence AA, p6
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