Mapping nitro-tyrosine modifications in fibrinogen by mass spectrometry as a biomarker for inflammatory disease

Stuart Meredith, Gita Parekh, Joanna Towler, James Schouten, Paul Davis, Helen Griffiths, Corinne Spickett

Research output: Contribution to journalMeeting abstract

Abstract

There is a growing awareness that inflammatory diseases have an oxidative pathology, which can result in specific oxidation of amino acids within proteins. It is known that patients with inflammatory disease have higher levels of plasma protein nitro-tyrosine than healthy controls. Fibrinogen is an abundant plasma protein, highly susceptible to such oxidative modifications, and is therefore a potential marker for oxidative protein damage. The aim of this study was to map tyrosine nitration in fibrinogen under oxidative conditions and identify susceptible residues. Fibrinogen was oxidised with 0.25mM and 1mM SIN-1, a peroxynitrite generator, and methionine was used to quench excess oxidant in the samples. The carbonyl assay was used to confirm oxidation in the samples. The carbonyl levels were 2.3, 8.72 and 11.5nmol/mg protein in 0, 0.25mM and 1mM SIN-1 samples respectively. The samples were run on a SDS-PAGE gel and tryptically digested before analysis by HPLC MS-MS. All 3 chains of fibrinogen were observed for all treatment conditions. The overall sequence coverage for fibrinogen determined by Mascot was between 60-75%. The oxidised samples showed increases in oxidative modifications in both alpha and beta chains, commonly methionine sulfoxide and tyrosine nitration, correlating with increasing SIN-1 treatment. Tyrosines that were most susceptible were Tyr135 (tryptic peptide YLQEIYNSNNQK) and Tyr277 (tryptic peptide GGSTSYGTGSETESPR), but several other nitrated tyrosines were also identified with high confidence. Identification of these susceptible peptides will allow design of sequences-specific biomarkers of oxidative and nitrative damage to plasma protein in inflammatory conditions.

LanguageEnglish
Article numberP87
PagesS50
Number of pages1
JournalFree Radical Biology and Medicine
Volume75
Issue numberSuppl.1
DOIs
Publication statusPublished - Oct 2014

Fingerprint

Biomarkers
Fibrinogen
Mass spectrometry
Tyrosine
Mass Spectrometry
Nitration
Blood Proteins
Peptides
Oxidation
Proteins
Peroxynitrous Acid
Pathology
Oxidants
Methionine
Polyacrylamide Gel Electrophoresis
Assays
Gels
High Pressure Liquid Chromatography
Amino Acids
Therapeutics

Cite this

Meredith, Stuart ; Parekh, Gita ; Towler, Joanna ; Schouten, James ; Davis, Paul ; Griffiths, Helen ; Spickett, Corinne. / Mapping nitro-tyrosine modifications in fibrinogen by mass spectrometry as a biomarker for inflammatory disease. In: Free Radical Biology and Medicine. 2014 ; Vol. 75, No. Suppl.1. pp. S50.
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abstract = "There is a growing awareness that inflammatory diseases have an oxidative pathology, which can result in specific oxidation of amino acids within proteins. It is known that patients with inflammatory disease have higher levels of plasma protein nitro-tyrosine than healthy controls. Fibrinogen is an abundant plasma protein, highly susceptible to such oxidative modifications, and is therefore a potential marker for oxidative protein damage. The aim of this study was to map tyrosine nitration in fibrinogen under oxidative conditions and identify susceptible residues. Fibrinogen was oxidised with 0.25mM and 1mM SIN-1, a peroxynitrite generator, and methionine was used to quench excess oxidant in the samples. The carbonyl assay was used to confirm oxidation in the samples. The carbonyl levels were 2.3, 8.72 and 11.5nmol/mg protein in 0, 0.25mM and 1mM SIN-1 samples respectively. The samples were run on a SDS-PAGE gel and tryptically digested before analysis by HPLC MS-MS. All 3 chains of fibrinogen were observed for all treatment conditions. The overall sequence coverage for fibrinogen determined by Mascot was between 60-75{\%}. The oxidised samples showed increases in oxidative modifications in both alpha and beta chains, commonly methionine sulfoxide and tyrosine nitration, correlating with increasing SIN-1 treatment. Tyrosines that were most susceptible were Tyr135 (tryptic peptide YLQEIYNSNNQK) and Tyr277 (tryptic peptide GGSTSYGTGSETESPR), but several other nitrated tyrosines were also identified with high confidence. Identification of these susceptible peptides will allow design of sequences-specific biomarkers of oxidative and nitrative damage to plasma protein in inflammatory conditions.",
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Mapping nitro-tyrosine modifications in fibrinogen by mass spectrometry as a biomarker for inflammatory disease. / Meredith, Stuart; Parekh, Gita; Towler, Joanna; Schouten, James; Davis, Paul; Griffiths, Helen; Spickett, Corinne.

In: Free Radical Biology and Medicine, Vol. 75, No. Suppl.1, P87, 10.2014, p. S50.

Research output: Contribution to journalMeeting abstract

TY - JOUR

T1 - Mapping nitro-tyrosine modifications in fibrinogen by mass spectrometry as a biomarker for inflammatory disease

AU - Meredith, Stuart

AU - Parekh, Gita

AU - Towler, Joanna

AU - Schouten, James

AU - Davis, Paul

AU - Griffiths, Helen

AU - Spickett, Corinne

PY - 2014/10

Y1 - 2014/10

N2 - There is a growing awareness that inflammatory diseases have an oxidative pathology, which can result in specific oxidation of amino acids within proteins. It is known that patients with inflammatory disease have higher levels of plasma protein nitro-tyrosine than healthy controls. Fibrinogen is an abundant plasma protein, highly susceptible to such oxidative modifications, and is therefore a potential marker for oxidative protein damage. The aim of this study was to map tyrosine nitration in fibrinogen under oxidative conditions and identify susceptible residues. Fibrinogen was oxidised with 0.25mM and 1mM SIN-1, a peroxynitrite generator, and methionine was used to quench excess oxidant in the samples. The carbonyl assay was used to confirm oxidation in the samples. The carbonyl levels were 2.3, 8.72 and 11.5nmol/mg protein in 0, 0.25mM and 1mM SIN-1 samples respectively. The samples were run on a SDS-PAGE gel and tryptically digested before analysis by HPLC MS-MS. All 3 chains of fibrinogen were observed for all treatment conditions. The overall sequence coverage for fibrinogen determined by Mascot was between 60-75%. The oxidised samples showed increases in oxidative modifications in both alpha and beta chains, commonly methionine sulfoxide and tyrosine nitration, correlating with increasing SIN-1 treatment. Tyrosines that were most susceptible were Tyr135 (tryptic peptide YLQEIYNSNNQK) and Tyr277 (tryptic peptide GGSTSYGTGSETESPR), but several other nitrated tyrosines were also identified with high confidence. Identification of these susceptible peptides will allow design of sequences-specific biomarkers of oxidative and nitrative damage to plasma protein in inflammatory conditions.

AB - There is a growing awareness that inflammatory diseases have an oxidative pathology, which can result in specific oxidation of amino acids within proteins. It is known that patients with inflammatory disease have higher levels of plasma protein nitro-tyrosine than healthy controls. Fibrinogen is an abundant plasma protein, highly susceptible to such oxidative modifications, and is therefore a potential marker for oxidative protein damage. The aim of this study was to map tyrosine nitration in fibrinogen under oxidative conditions and identify susceptible residues. Fibrinogen was oxidised with 0.25mM and 1mM SIN-1, a peroxynitrite generator, and methionine was used to quench excess oxidant in the samples. The carbonyl assay was used to confirm oxidation in the samples. The carbonyl levels were 2.3, 8.72 and 11.5nmol/mg protein in 0, 0.25mM and 1mM SIN-1 samples respectively. The samples were run on a SDS-PAGE gel and tryptically digested before analysis by HPLC MS-MS. All 3 chains of fibrinogen were observed for all treatment conditions. The overall sequence coverage for fibrinogen determined by Mascot was between 60-75%. The oxidised samples showed increases in oxidative modifications in both alpha and beta chains, commonly methionine sulfoxide and tyrosine nitration, correlating with increasing SIN-1 treatment. Tyrosines that were most susceptible were Tyr135 (tryptic peptide YLQEIYNSNNQK) and Tyr277 (tryptic peptide GGSTSYGTGSETESPR), but several other nitrated tyrosines were also identified with high confidence. Identification of these susceptible peptides will allow design of sequences-specific biomarkers of oxidative and nitrative damage to plasma protein in inflammatory conditions.

U2 - 10.1016/j.freeradbiomed.2014.10.819

DO - 10.1016/j.freeradbiomed.2014.10.819

M3 - Meeting abstract

VL - 75

SP - S50

JO - Free Radical Biology and Medicine

T2 - Free Radical Biology and Medicine

JF - Free Radical Biology and Medicine

SN - 0891-5849

IS - Suppl.1

M1 - P87

ER -