Evaluation of Evaluation of “Top“Top--down” and “Bottomdown” and “Bottom--up” up”

Approaches to the Identification of Approaches to the Identification of Mutations in Atlantic Cod Mutations in Atlantic Cod HemoglobinsHemoglobins

Wendell P. Griffith; Joshua K. Hoerner; and Igor A. KaltashovUniversity of Massachusetts, Amherst, MA

OverviewOverview

• Molecular masses calculated from ESI mass spectra for the α- and β-chains of Atlantic cod hemoglobin are inconsistent with the masses calculated from their published sequences.

• “Top-down” and “Bottom-up” approaches were used in an attempt to identify the mutations responsible for the discrepancies in mass.

• Either approach provides much information about the sites of mutation in the protein. For complete mutation analysis of cod hemoglobins though, both approaches must be used.

IntroductionIntroduction• The significance of biochemical genetic variations of fishes is

often unclear, though such variations have been studied for about 4 decades.

• Atlantic cod (Gadus morhua) hemoglobins (Hb-I) show great polymorphism with the genotypes Hb-I(1/1), Hb-I(1/2), Hb-I(2/2), Hb-I(1/2b), and Hb-I(2/2b) which all differ in O2 affinity.

• The phenotype expressed is dependent on the fishes’location along the northern and western Norwegian and the North American east coastline.

• Even though such great phenotypic diversity is known, there is only one published sequence (α: O42425 and β:O13077) for either chain of Atlantic cod hemoglobin.

ExperimentalExperimental

• Mass Spectrometry:• 4.7T Apex III (Bruker Daltonics, Billerica, MA) FT ICR

MS equiped with a standard ESI source• Esquire-LC QIT MS (Bruker Daltonics, Billerica, MA)

• Liquid Chromatography:• Agilent 1100 Series HPLC (Agilent Technologies,

USA)

• Sample Preparation:• Hemolysate was isolated from Atlantic cod collected

form the Massachusetts coastline at Gloucester.• Proteomics grade TPCK-treated trypsin used for

proteolysis

DeconvolutionDeconvolution

α-chain

•15829.8 (∆ = + 45.8)

β-chain

•16664.5 (∆ = + 82.5)

ESIESI--MS of Atlantic Cod MS of Atlantic Cod HemolysateHemolysate

50:47:3 methanol, H2O, acetic acid

BottomBottom--up Approach: Digestion of up Approach: Digestion of HemolysateHemolysate

Gadus morhua hemolysate incubated with trypsin for 40 hrs at 37°C.Digestion carried out in 100 mM AmBic buffer, pH 8.5.

P Y V A V I/L

Q T K

αα3333--4141

Expected mass:1018.59371018.5937

LVAVYPQTKLVAVYPQTK

Example of ESI Example of ESI QIT MSQIT MS22

MSLSSKQKAT VKDFFSKMST RSDDIGAEALSRLVAVYPQT KSYFSHWKDA SPGSAPVRKHGITTMGGVYD AVGKIDDLKG GLLSLSELHAFMLRVDPVNF KLLAHCMLVC MSMIFPEEFTPQVHVAVDKF LAQLALALAE KYR

VEWTDSERAI INSIFSNLDY EEIGRKSLCRCLIVYPWTQR YFGGFGNLYN AETILCNPLIAAHGTKILHG LDRALKNMDD IKNTYAELSLLHSDKLHVDP DNFRLLADCL TGVIAAKMVPAFTVDTQVGW QKFRSFVVSA LGREYH

HBA GADMO

HBB GADMO

Identified by Mass & MS/MS Identified by Mass only

Summary: “BottomSummary: “Bottom--up” Approachup” Approach

Chain IsolationChain Isolation

0

10

20

30

40

0 5 10 15 20 25 30 35 40 45Time (min)

mA

U

Gadus morhua Hemolysate

Polaris C18-A, 3uM, 4.6x250mm214 nm, 1.0 mL/minA: 0.1% TFA in 5% ACNB: 0.1% TFA in 95% ACNGradient: Iso at 45% B for 15 min, 45 - 50% B in 30 min, 50% B for 15 min

11.5

78

23.3

65

43.5

67

40.7

05

36.1

84α: 43.567 min

β: 40.705 min

Identification of Identification of Globin Globin FractionsFractions

Fraction at 43.567 min

Fraction at 40.705 minβ- chain

α- chain

TopTop--down: down: αα--chainchain

Correct Correct SequenceSequence

Mutated Mutated SequenceSequence

Ladder of +10charge state ions

1160 1170 1180 1190 1200 1210 1220 1230 1240 1250 1260

TopTop--down: down: ββ--chainchain

Correct Correct SequenceSequence

Mutated Mutated SequenceSequence

MSLSSKQKAT VKDFFSKMST RSDDIGAEALSRLVAVYPQT KSYFSHWKDA SPGSAPVRKHGITTMGGVYD AVGKIDDLKG GLLSLSELHAFMLRVDPVNF KLLAHCILVV MAIMFPEEFTPQVHVAVDKF LAQLALALAE KXKYR

VEWTDSERAI INSIFSNLDY EEIGRKSLCRCLIVYPWTQR YFGGFGNLYN AETIMALCNP LIAAHGTKIL HGLDRALKNM DDIKNTYAEL SLLHSDKLHV DPDNFRLLAD CLTGVIAAKM VPAFTVDTQV GWQKFLSVVV SALGREYH

HBA GADMO

HBB GADMO

Summary: “TopSummary: “Top--down” Approachdown” Approach

Mutation Sequence InsertionSequence Confirmed

Database Search for MutationsDatabase Search for Mutations

• Search sequences: [I/L][I/L]VVMA[I/L]M and M[I/L]AMVV[I/L] [I/L]

• Matching sequences:• [ILVVMAIM] Hemoglobin Alpha from Japanese rice fish• [ILVVMAIM] Hemoglobin Alpha from Spotted wolffish

• Search sequences:KF[I/L]SVVVSA and ASVVVS[I/L]FK

• Matching sequences:• [KFLSVVVSA] Hemoglobin Beta from Five-ray yellowtail• [KFLSVVVSA] Hemoglobin Beta from Atlantic salmon• [KFLSVVVSA] Hemoglobin Beta from Rainbow trout• [KFLSVVVSA] Hemoglobin Beta from Grass Carp• [KFLSVVVSA] Hemoglobin Beta from Zebra fish• [KFLSVVVSA] Hemoglobin Beta from Goldfish

Comparative Mutation AnalysisComparative Mutation AnalysisMSLSSKQKAT VKDFFSKMST RSDDIGAEAL SRLVAVYPQT MSLSAKDKAA VKAFWAKVSG QADAIGSDAL SRMLVVYPQTSLTAKDKDT VRAFWAKASG KAAEIGSDAL SRMLVVYPQT

KSYFSHWKDA SPGSAPVRKH GITTMGGVYD AVGKIDDLKGKTYFAHWKDL SPGSAPVKKH GKTVMGGIAD AVGKIDDISSKTYFSHWKDT SPGSEPVKKH GKSVMGGVAD AVMKIEDLNA

GLLSLSELHA FMLRVDPVNF KLLAHCMLVC MSMIFPEEFT GLLNLSELHA FTLRVDPTNF KILSHNILVV MAIMFPQDFTGLLNLSELHA FTLRVDPANF KILSHNILVV MAIMFPKDFT

PQVHVAVDKF LAQLALALAE KYRPEVHVALDKF LAAVSLALSE KYRPEVHVAMDFK LAALSRALAE KYR

Atlantic cod

Japanese rice fish

Artic spotted wolffish

Possible mutation based on sequence comparison

MSLSSKQKAT VKDFFSKMST RSDDIGAEALSRLVAVYPQT KSYFSHWKDA SPGSAPVRKHGITTMGGVYD AVGKIDDLKG GLLSLSELHAFMLRVDPVNF KLLAHCILVV MAIMFPEEFTPQVHVAVDKF LAQLALALAE KXKYR

VEWTDSERAI INSIFSNLDY EEIGRKSLCRCLIVYPWTQR YFGGFGNLYN AETIMALCNP LIAAHGTKIL HGLDRALKNM DDIKNTYAEL SLLHSDKLHV DPDNFRLLAD CLTGVIAAKM VPAFTVDTQV GWQKFLSVVV SALGREYH

HBA GADMO

HBB GADMO

Mutation Sequence Insertion

Summary of all DataSummary of all Data

Sequence Confirmed

ConclusionsConclusions• Both the “top-down” and “bottom-up” approaches to

sequence analysis were useful in elucidating correct and mutated parts of the sequence of Atlantic cod hemoglobin.

• Due to the vast number of mutations, for complete and expeditious mapping of the mutated parts of the sequence, a combination of both approaches is required.

• Although not a de novo sequencing exercise, this research will aid in future timely identification of the sites of modification and mutation in fish hemoglobins. It may even provide a method of cataloging specimens based on their locale.

• Some additional work is needed to complete this project.

AcknowledgementsAcknowledgements

• Prof. Herbert O. Hultin– UMass Marine Station

• Stephen J. Eyles

• Joshua K. Hoerner

• University of Massachusetts, Department of Chemistry

ReferencesReferencesBrix, O., Forås, E., and Strand, I. (1998) Comp. Biochem. Physiol. 119A, 575 - 583.

Sick, K. (1961) Nature 192, 894-896.

Frydenberg, O., Møller, D., Nævdal, G., and Sick, K. (1965) Hereditas. 53, 257-271.

Sick, K. (1965) Hereditas. 54, 19-48.

Sick, K. (1965) Hereditas. 54, 49-68.

Swiss-PROT sequences from nucleic acid O42425, O13077.