Quantitation of Changes in Protein Expression between Chicken Dark and White Meat
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Quantitation of Changes in Protein Expression between Chicken Dark and White Meat by PMF and MSMS. B. MYH peptides. MYH peptides. w. w. w. d1. d1. d1. d2. d2. d2. d1. d1. d3. d3. d3. wd1. wd1. wd1. wd2. wd2. wd2. d1d2. d1d2. d1d2. w. w. wd1d2. wd1d2. wd1d2.

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Other enzymes

Quantitation of Changes in Protein Expression between Chicken Dark and White Meat

by PMF and MSMS.

B.

MYH peptides

MYH peptides

w

w

w

d1

d1

d1

d2

d2

d2

d1

d1

d3

d3

d3

wd1

wd1

wd1

wd2

wd2

wd2

d1d2

d1d2

d1d2

w

w

wd1d2

wd1d2

wd1d2

LC separation; TIC vs fraction #

Parent spectrum #649

PC2

1869 peak is 5th highest

d2

d2

MS spectrum of unseparated chicken dark meat digest

Other Enzymes

Other Enzymes

Other Enzymes

glycolytic

glycolytic

glycolytic

w

HSPB2

HSPB2

HSPB2

850

3200

CK

CK

CK

d1

d2

PCA2

PCA2

PCA1

PCA1

PC1

MSMS spectrum of aSSDAEMAIFGEAAPYLR, Mascot score=56

1820

1920

1869 peak is the highest

Parent spectrum #461

b4

b6

b5

y14

150

1650

PSD-like msms spectrum of 1869 isotope cluster

y6

b6

MSMS spectrum of DLIELQALIDSHFEAR, Mascot score=95

b7

b2

H

L

b8

1

b5

y6

1596

y4

y3

b4

60

10

Fig. 5. MS-MS spectrum of SYELPDGQVITIGNER from actin (strongest peak) from whole digest using SimulTof 300 tandem. Mascot Score 104.

Intensity

Intensity

50

14 samples:

6 white meat

8 dark meat

8

40

6

b2

30

y6

y6

4

20

b4

2

10

w,v9

0

0

2

4

6

8

12

2

4

6

8

0

10

14

0

10

12

14

Sample Number

Sample Number

1384.74

1384.74

ANLLQAE

ANLLQAE

V

V

EELR

EELR

1283

1681

100 Most intense masses from each

1398.76

1398.76

ANLLQAE

ANLLQAE

I

I

EELR

EELR

1386.72

1386.72

ANLLQAE

ANLLQAE

T

T

EELR

EELR

7

20

Intensity

6

Intensity

15

5

dark

white

4

10

Merged mass list with 226 masses and intensities

3

2

5

1

0

0

0

4

6

8

0

6

2

10

12

14

2

4

8

10

12

14

Sample Number

Sample Number

y10

1373.69

1373.69

VAEQEL

VAEQEL

L

L

DA

DA

T

T

ER

ER

1704

1424

1359.68

1359.68

VAEQEL

VAEQEL

L

L

DA

DA

S

S

ER

ER

14

10

ib4

PDGQ

1377.63

1377.63

VAEQEL

VAEQEL

M

M

DA

DA

S

S

ER

ER

Intensity

Intensity

12

ib5

PDGQV

8

y3

ib6

PDGQVI

10

6

8

b9

y12

y13

b2

y7

w13

b8

6

4

4

2

2

0

0

2

4

8

0

2

4

6

8

10

0

12

14

6

10

12

14

Sample Number

Sample Number

WP20 #411

Kenneth C. Parker, Stephen J. Hattan, Marvin Vestal.SimulTof Corporation, Sudbury , MA

Fig. 4. Comparison of MS and MS-MS spectra obtained via LC-MALDI vs spectra obtained directly from separated digest focusing on mass 1869. 8.

Introduction

Fig. 1. PCA analysis

Fig. 3. Same region following LC separation. High energy msms spectra.

B.

MYH peptides

MYH peptides

Many multicellular organisms contain several different kinds of muscle fibers, which result from the expression of distinct combinations of major muscle protein genes. Muscles are classifiable as fast or slow, based predominantly on myosin heavy chain genes. Many other major muscle proteins derive from a family of homologous genes. In humans, myosin heavy chain usage changes with disease, with muscle type and according to exercise patterns. Moreover, muscle preparations tend to contain a mixed population of slow twitch and fast twitch muscle. Chicken was chosen as a model organism because meat is readily available enriched in slow fibers (dark meat or fast fibers (white meat). We have determined how major muscle proteins differ across meat types.

w

w

w

Sample-based PCA

d1

d1

d1

d2

d2

d2

d1

d1

d3

d3

d3

wd1

wd1

wd1

wd2

wd2

wd2

d1d2

d1d2

d1d2

w

w

A.

wd1d2

wd1d2

wd1d2

PC2

d2

d2

PC1

Same spectrum, inset 1

C.

C.

3 distinct categories

PC2

Same spectrum, inset 2

Fig1B,C. Mass-based PCA

Methods

MYH: masses mapped to any myosin heavy chain isoform. In mass-based PCA, the strongest drivers correspond to MYH, though some MYH masses (in black in Fig.1B) are constant. Most glycolytic enzyme masses are strongest in white meat, while creatine kinase (CK) is enriched in both dark meat categories. Heat shock protein HSPB2 is enriched in the dark meat category ‘d1’.

Meat samples were homogenized, reduced, alkylated, and digested with trypsin. Digests were subjected directly to PMF, resulting in the identification of tens of proteins. In addition, the same digest preparations were subjected to HPLC-MALDI analysis, and parent MS spectra and MS-MS spectra were acquired. Each separation resulted in a peak list containing ~ 10000 peptide masses, measured with < 5 ppm accuracy with the use of two internal standards. PMF software was used that takes into account preferential arginine detection, and novel MS-MS software was developed to interpret high energy MS-MS spectra. The intensities from the same peak lists allowed quantitation of changes between muscle types. In the last few weeks, MS-MS spectra were gathered directly using SimulTof 300 Tandem.

1867

1876

Fig 2. Distribution of selected masses over the 14 samples

Daughter ions map to both DLIELQALIDSHFEAR (troponin; mz 1869.9711) and aSSDAEMAIFGEAAPYLR (MYH N-terminus; mz 1869.8694). Much of the ‘ noise’ shows every mz fine structure, presumably deriving from other precursors.

  • Conclusions:

  • Using MS, can see differences between meat samples.

  • Using PCA, one can separate samples into 3 categories.

    • White meat, two separate dark meat categories.

  • Can identify 5-10 proteins from chicken muscle by PMF.

  • Confirmation of PMF by MSMS can be performed directly on unseparated digests, resulting in ~15 protein IDs from whole chicken muscle so far.

  • More confirmation obtained by MSMS following LC-MALDI.

  • Differences correspond to MYH isoforms, glycolytic enzymes, etc.

  • References:

  • 1.) Parker KC. Scoring Methods in MALDI Peptide Mass Fingerprinting:

  • ChemScore and the ChemApplex Program. JASMS 2002;13:22-39.

  • Funding:SBIR grant 1R44GM090389-01A1.

Workflows:

  • Perform PMF on tryptic digest of whole tissue.

  • Perform PCA on mass lists

  • 3. Acquire MS-MS spectra using LC-MALDI

  • 4. Collect MS-MS spectra directly from unseparated digests

Amino acids that are variable between myosin isoforms are color-coded as on Fig. 1. Some peptides have only 2 forms.

Many other MYH peptides are shared between these isoforms.

Peptides aligned to one MYH isoform, with 1st aa listed.


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