Chemically-Assisted Fragmentation Combined with Multi-Dimensional Liquid Chromatography and Matrix-Assisted Laser Desorption/Ionization Post Source Decay,Matrix-Assisted Laser Desorption/Ionization Tandem Time-of-Flight or Matrix-Assisted Laser Desorption/Ionization Tandem Mass Spectrometry for Improved Sequencing of Tryptic Peptides

Abstract

Derivatization of tryptic peptides using an Ettan CAF matrix-assisted laser desorption/ionization (MALDI) sequencing kit in combination with MALDI-post source decay (PSD) is a fast, accurate and convenient way to obtain de novo or confirmative peptide sequencing data. CAF (chemically assisted fragmentation) is based on solid-phase derivatization using a new class of water stable sulfonation agents, which strongly improves PSD analysis and simplifies the interpretation of acquired spectra. The derivatization is performed on solid supports, ZipTip_μC18, limiting the maximum peptide amount to 5 μg. By performing the derivatization in solution enabled the labeling of tryptic peptides derived from 100 μg of protein. To increase the number of peptides that could be sequenced, derivatized peptides were purified using multidimensional liquid chromatography (MDLC) prior to MALDI sequencing. Following the first dimension strong cation exchange (SCX) chromatography step, modified peptides were separated using reversed-phase chromatography (RPC). During the SCX clean up step, positively charged peptides are retained on the column while properly CAF-derivatized peptides (uncharged) are not. A moderately complex tryptic digest, prepared from six different proteins of equimolar amounts, was CAF-derivatized and purified by MDLC. Fractions from the second dimension nano RPC step were automatically sampled and on-line dispensed to MALDI sample plates and analyzed using MALDI mass spectrometry fragmentation techniques. All proteins in the derivatized protein mixture digest were readily identified using MALDI-PSD or MALDI tandem mass spectrometry (MS/MS). More than 40 peptides were unambiguously sequenced, representing a seven-fold increase in the number of sequenced peptides in comparison to when the CAF-derivatized protein mix digest was analyzed directly (no MDLC-separation) using MALDI-PSD. In conclusion, MDLC purification of CAF-derivatized peptides significantly increases the success rate for de novo and confirmative sequencing using various MALDI fragmentation techniques. This new approach is not only applicable to single protein digests but also to more complex digests and could, thus, be an alternative to electrospray ionization MS/MS for peptide sequencing.

Keywords

MALDI-ToF MS MALDI-PSD CAF derivatization chemically assisted fragmentation MDLC peptide sequencing

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References

Keough

Lacey

M.P.

Youngquist

R.S.

, “A method for high-sensitivity peptide sequencing using postsource decay matrix-assisted laser desorption ionization mass spectrometry,” Proc. Natl. Acad. Sci. USA 96, 7131 (1999).

Spengler

, “Post-source decay analysis in matrix-assisted laser desorption/ionization mass spectrometry of biomolecules”, J. Mass Spectrom. 32, 1019 (1997).

Bauer

M.D.

Sun

Keough

Lacey

M.P.

, “Sequencing of sulfonic acid derivatized peptides by electrospray mass spectrometry”, Rapid Commun. Mass Spectrom. 14, 924 (2000).

Liminga

Carlsson

Larsson

Maloisel

J.L.

Palmgren

Keough

Youngquist

R.S.

, Proc 49^th ASMS Conference on Mass Spectrometry and Allied Topics, Chicago, IL, USA (2001).

Keough

Lacey

M.P.

Youngquist

R.S.

, “Solid-phase derivatization of tryptic peptides for rapid protein identification by matrix-assisted laser desorption/ionization mass spectrometry”, Rapid Commun. Mass Spectrom. 16, 1003 (2002).

Hellman

Bhikhabhai

, “Easy amino acid sequencing of sulfonated peptides using post-source decay on a matrix-assisted laser desorption/ionization time-of-flight spectrometer equipped with a variable voltage reflector”, Rapid Commun. Mass Spectrom. 16, 1851 (2002).

Flensburg

Belew

, “Characterization of recombinant human serum albumin using matrix-assisted laser desorption ionization time-of-flight mass spectrometry”, J. Chromatogr. A 1009, 111 (2003).

Eklund

Andersson

H.O.

Kamali-Moghaddam

Sundström

Flensburg

, “Purification and partial characterization by matrix-assisted laser desorption ionization time-of-flight mass spectrometry of the recombinant transposase, TniA”, J. Chromatogr. A 1009, 179 (2003).

Flensburg

Haid

Blomberg

Bielawski

Ivansson

, “Applications and performance of a MALDI-ToF mass spectrometer with quadratic field reflectron technology”, J. Biochem. Biophys. Methods 60, 319 (2003).

10.

Keough

Lacey

M.P.

Youngquist

R.S.

, “Derivatization procedures to facilitate de novo sequencing of lysine-terminated tryptic peptides using postsource decay matrix-assisted laser desorption/ionization mass spectrometry”, Rapid Commun. Mass Spectrom. 14, 2348 (2000).

11.

Wysocki

V.H.

Tsaprailis

Smith

L.L.

Breci

L.A.

, “Mobile and localized protons: A framework for understanding peptide dissociation”, J. Mass Spectrom. 35, 1399 (2000).

12.

Shevchenko

Jensen

O.N.

Podtelejnokov

Sagliocco

Wilm

Vorm

Mortensen

Shevchenko

Boucherie

Mann

, “Linking genome and proteome by mass spectrometry: Large-scale identification of yeast proteins from two dimensional gels”, Proc. Natl. Acad. Sci. USA 93, 1440 (1996).

13.

Zhang

Chait

B.T.

, “ProFound: An expert system for protein identification using mass spectrometric peptide mapping information”, Anal. Chem. 72, 2482 (2000).

14.

Field

H.I.

Fenyö

Beavis

R.C.

, “RADARS, a bio-informatics solution that automates proteome mass spectral analysis, optimises protein identification, and archives data in a relational database”, Proteomics 2, 36 (2002).