Fourier-transform mass spectrometry for detection of thioester-bound intermediates in unfractionated proteolytic mixtures of 80 and 191 kDa portions of Bacitracin A synthetase

Leslie Hicks; Paul Weinreb; Dirk Konz; Mohamed A. Marahiel; Christopher T. Walsh; Neil L. Kelleher

doi:10.1016/S0003-2670(03)01001-8

Fourier-transform mass spectrometry for detection of thioester-bound intermediates in unfractionated proteolytic mixtures of 80 and 191 kDa portions of Bacitracin A synthetase

Leslie Hicks, Paul Weinreb, Dirk Konz, Mohamed A. Marahiel, Christopher T. Walsh, Neil L. Kelleher^*

^*Corresponding author for this work

Molecular Biosciences

Research output: Contribution to journal › Article › peer-review

7 Scopus citations

Abstract

Enzymes involved in polyketide and non-ribosomal peptide biosynthesis utilize covalent intermediates on several carrier sites along their exceptionally large primary structures (100-1000kDa). Direct detection of these thioester-bound intermediates using mass spectrometry (MS) could dissect the timing of intermediate formation and translocation. Proteolysis of BacA1 (80kDa), the first module in the production of Bacitracin A, using Endoproteinase Glu-C yielded 202 peptides from 1 to 20kDa as detected using electrospray ionization with Fourier-transform MS at 9.4T. A 1:1 mixture of Ile and ¹³C₆-Ile allowed the selective detection of the Ile-thioester bound intermediate directly from the 200-component mixture. Direct FTMS analysis of the Glu-C digestion products from the two module BacA1-2 construct (191kDa) gave perhaps the most complicated spectrum of a proteolytic digest ever recorded at isotopic resolution. This 9.4T spectrum (1000 scans) contains 759 individual isotopic distributions from 528 peptides from 1 to 30kDa. While the Glu-C product from the BacA1 carrier site was again detected, a peptide corresponding to the second carrier site was not. This indicates that even using the highest resolution type of mass spectrometry, some peptide fractionation (e.g., using capillary chromatography) will be required to detect thioester-bound intermediates in a robust fashion from such large "assembly line" catalysts.

Original language	English (US)
Pages (from-to)	217-224
Number of pages	8
Journal	Analytica Chimica Acta
Volume	496
Issue number	1-2
DOIs	https://doi.org/10.1016/S0003-2670(03)01001-8
State	Published - Oct 31 2003

Funding

We are grateful to Chris Hendrickson, John Quinn, Mark Emmett, and Alan Marshall for access to a 9.4 Tesla FTMS (National High-Field FT-ICR MS facility, NHMFL, NSF CHE-94-13008). We further acknowledge the generous support from the University of Illinois and a NSF Fellowship to LH. CTW acknowledges the National Institutes of Health (GM20011) and MM was supported by a grant from the Deutsche Forschungsgemeinschaft. Professor Kelleher received a B.S. and B.A. from Pacific Lutheran U. in 1992, a Fulbright Fellowship the following year, and a Ph.D. with Tadhg Begley and Fred McLafferty (Cornell U.) in 1997. After a NIH Postdoctoral Fellowship with Chris Walsh (Harvard Medical School), Kelleher joined the faculty at the University of Illinois in 1999. He has received several awards including a Packard Fellowship, the NSF CAREER Award, the Lilly Analytical Chemistry Award, and support from the Burroughs Wellcome, the Searle, and the Dreyfus Foundations. Kelleher has interest in Mass Spectrometry-based enzymology and “Top Down” proteomics using intact proteins for efficient detection of their post-translational modifications.

Keywords

Acyl enzyme
Covalent modification
Electrospray
Fourier-transform mass spectrometry
NRPS
Stable isotope

ASJC Scopus subject areas

Analytical Chemistry
Biochemistry
Environmental Chemistry
Spectroscopy

Access to Document

10.1016/S0003-2670(03)01001-8

Cite this

@article{87ddc52e73ac4f9d8e5ba8e3b2d97c88,

title = "Fourier-transform mass spectrometry for detection of thioester-bound intermediates in unfractionated proteolytic mixtures of 80 and 191 kDa portions of Bacitracin A synthetase",

abstract = "Enzymes involved in polyketide and non-ribosomal peptide biosynthesis utilize covalent intermediates on several carrier sites along their exceptionally large primary structures (100-1000kDa). Direct detection of these thioester-bound intermediates using mass spectrometry (MS) could dissect the timing of intermediate formation and translocation. Proteolysis of BacA1 (80kDa), the first module in the production of Bacitracin A, using Endoproteinase Glu-C yielded 202 peptides from 1 to 20kDa as detected using electrospray ionization with Fourier-transform MS at 9.4T. A 1:1 mixture of Ile and 13C6-Ile allowed the selective detection of the Ile-thioester bound intermediate directly from the 200-component mixture. Direct FTMS analysis of the Glu-C digestion products from the two module BacA1-2 construct (191kDa) gave perhaps the most complicated spectrum of a proteolytic digest ever recorded at isotopic resolution. This 9.4T spectrum (1000 scans) contains 759 individual isotopic distributions from 528 peptides from 1 to 30kDa. While the Glu-C product from the BacA1 carrier site was again detected, a peptide corresponding to the second carrier site was not. This indicates that even using the highest resolution type of mass spectrometry, some peptide fractionation (e.g., using capillary chromatography) will be required to detect thioester-bound intermediates in a robust fashion from such large {"}assembly line{"} catalysts.",

keywords = "Acyl enzyme, Covalent modification, Electrospray, Fourier-transform mass spectrometry, NRPS, Stable isotope",

author = "Leslie Hicks and Paul Weinreb and Dirk Konz and Marahiel, {Mohamed A.} and Walsh, {Christopher T.} and Kelleher, {Neil L.}",

note = "Funding Information: We are grateful to Chris Hendrickson, John Quinn, Mark Emmett, and Alan Marshall for access to a 9.4 Tesla FTMS (National High-Field FT-ICR MS facility, NHMFL, NSF CHE-94-13008). We further acknowledge the generous support from the University of Illinois and a NSF Fellowship to LH. CTW acknowledges the National Institutes of Health (GM20011) and MM was supported by a grant from the Deutsche Forschungsgemeinschaft. Funding Information: Professor Kelleher received a B.S. and B.A. from Pacific Lutheran U. in 1992, a Fulbright Fellowship the following year, and a Ph.D. with Tadhg Begley and Fred McLafferty (Cornell U.) in 1997. After a NIH Postdoctoral Fellowship with Chris Walsh (Harvard Medical School), Kelleher joined the faculty at the University of Illinois in 1999. He has received several awards including a Packard Fellowship, the NSF CAREER Award, the Lilly Analytical Chemistry Award, and support from the Burroughs Wellcome, the Searle, and the Dreyfus Foundations. Kelleher has interest in Mass Spectrometry-based enzymology and “Top Down” proteomics using intact proteins for efficient detection of their post-translational modifications.",

year = "2003",

month = oct,

day = "31",

doi = "10.1016/S0003-2670(03)01001-8",

language = "English (US)",

volume = "496",

pages = "217--224",

journal = "Analytica Chimica Acta",

issn = "0003-2670",

publisher = "Elsevier B.V.",

number = "1-2",

}

TY - JOUR

T1 - Fourier-transform mass spectrometry for detection of thioester-bound intermediates in unfractionated proteolytic mixtures of 80 and 191 kDa portions of Bacitracin A synthetase

AU - Hicks, Leslie

AU - Weinreb, Paul

AU - Konz, Dirk

AU - Marahiel, Mohamed A.

AU - Walsh, Christopher T.

AU - Kelleher, Neil L.

N1 - Funding Information: We are grateful to Chris Hendrickson, John Quinn, Mark Emmett, and Alan Marshall for access to a 9.4 Tesla FTMS (National High-Field FT-ICR MS facility, NHMFL, NSF CHE-94-13008). We further acknowledge the generous support from the University of Illinois and a NSF Fellowship to LH. CTW acknowledges the National Institutes of Health (GM20011) and MM was supported by a grant from the Deutsche Forschungsgemeinschaft. Funding Information: Professor Kelleher received a B.S. and B.A. from Pacific Lutheran U. in 1992, a Fulbright Fellowship the following year, and a Ph.D. with Tadhg Begley and Fred McLafferty (Cornell U.) in 1997. After a NIH Postdoctoral Fellowship with Chris Walsh (Harvard Medical School), Kelleher joined the faculty at the University of Illinois in 1999. He has received several awards including a Packard Fellowship, the NSF CAREER Award, the Lilly Analytical Chemistry Award, and support from the Burroughs Wellcome, the Searle, and the Dreyfus Foundations. Kelleher has interest in Mass Spectrometry-based enzymology and “Top Down” proteomics using intact proteins for efficient detection of their post-translational modifications.

PY - 2003/10/31

Y1 - 2003/10/31

N2 - Enzymes involved in polyketide and non-ribosomal peptide biosynthesis utilize covalent intermediates on several carrier sites along their exceptionally large primary structures (100-1000kDa). Direct detection of these thioester-bound intermediates using mass spectrometry (MS) could dissect the timing of intermediate formation and translocation. Proteolysis of BacA1 (80kDa), the first module in the production of Bacitracin A, using Endoproteinase Glu-C yielded 202 peptides from 1 to 20kDa as detected using electrospray ionization with Fourier-transform MS at 9.4T. A 1:1 mixture of Ile and 13C6-Ile allowed the selective detection of the Ile-thioester bound intermediate directly from the 200-component mixture. Direct FTMS analysis of the Glu-C digestion products from the two module BacA1-2 construct (191kDa) gave perhaps the most complicated spectrum of a proteolytic digest ever recorded at isotopic resolution. This 9.4T spectrum (1000 scans) contains 759 individual isotopic distributions from 528 peptides from 1 to 30kDa. While the Glu-C product from the BacA1 carrier site was again detected, a peptide corresponding to the second carrier site was not. This indicates that even using the highest resolution type of mass spectrometry, some peptide fractionation (e.g., using capillary chromatography) will be required to detect thioester-bound intermediates in a robust fashion from such large "assembly line" catalysts.

AB - Enzymes involved in polyketide and non-ribosomal peptide biosynthesis utilize covalent intermediates on several carrier sites along their exceptionally large primary structures (100-1000kDa). Direct detection of these thioester-bound intermediates using mass spectrometry (MS) could dissect the timing of intermediate formation and translocation. Proteolysis of BacA1 (80kDa), the first module in the production of Bacitracin A, using Endoproteinase Glu-C yielded 202 peptides from 1 to 20kDa as detected using electrospray ionization with Fourier-transform MS at 9.4T. A 1:1 mixture of Ile and 13C6-Ile allowed the selective detection of the Ile-thioester bound intermediate directly from the 200-component mixture. Direct FTMS analysis of the Glu-C digestion products from the two module BacA1-2 construct (191kDa) gave perhaps the most complicated spectrum of a proteolytic digest ever recorded at isotopic resolution. This 9.4T spectrum (1000 scans) contains 759 individual isotopic distributions from 528 peptides from 1 to 30kDa. While the Glu-C product from the BacA1 carrier site was again detected, a peptide corresponding to the second carrier site was not. This indicates that even using the highest resolution type of mass spectrometry, some peptide fractionation (e.g., using capillary chromatography) will be required to detect thioester-bound intermediates in a robust fashion from such large "assembly line" catalysts.

KW - Acyl enzyme

KW - Covalent modification

KW - Electrospray

KW - Fourier-transform mass spectrometry

KW - NRPS

KW - Stable isotope

UR - http://www.scopus.com/inward/record.url?scp=0242266935&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0242266935&partnerID=8YFLogxK

U2 - 10.1016/S0003-2670(03)01001-8

DO - 10.1016/S0003-2670(03)01001-8

M3 - Article

AN - SCOPUS:0242266935

SN - 0003-2670

VL - 496

SP - 217

EP - 224

JO - Analytica Chimica Acta

JF - Analytica Chimica Acta

IS - 1-2

ER -

Fourier-transform mass spectrometry for detection of thioester-bound intermediates in unfractionated proteolytic mixtures of 80 and 191 kDa portions of Bacitracin A synthetase

Abstract

Funding

Keywords

ASJC Scopus subject areas

Access to Document

Other files and links

Fingerprint

Cite this