C. Other Enzymes

PCA1

PC

A2

glycolytic

HSPB2

CK

Other Enzymes

PCA1

PC

A2

Other Enzymes

PC1

glycolytic

HSPB2

CK

glycolytic

HSPB2

CK

Quantitation of Changes in Protein Expression between Chicken Dark and White Meat by PMF and MSMS.

WP20 #411Kenneth C. Parker, Stephen J. Hattan, Marvin Vestal. SimulTof Corporation, Sudbury , MA

Methods 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.

IntroductionMany 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.

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.

1. Perform PMF on tryptic digest of whole tissue.2. Perform PCA on mass lists3. Acquire MS-MS spectra using LC-MALDI4. Collect MS-MS spectra directly from unseparated digests

wa

wb

wc

wd

wewf

d1a d1b

d2a

d2bd2c

d2d

d1c

d1d

PC1

PC

2

wa

wb

wc

wd

wewf

d1a d1b

d2a

d2bd2c

d2d

d1c

d1d

PC1

PC

2

14 samples:6 white meat8 dark meat

100 Most intense masses from each

Merged mass list with 226 masses and intensities

Fig. 1. PCA analysis

3 distinct categories

dark white

Workflows:

Sample-based PCA

Fig1B,C. Mass-based PCA

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’.

wd1d2

1

0102030405060

0 2 4 6 8 10 12 14Sample Number

Inte

nsi

ty

aSSDSEMAIFGEAAPYLR1885.86

aSSDAEMAIFGEAAPYLR1869.87

aASPDAEMAAFGEAAPYLR1908.88

aSSDSEMAIFGEAAPYLR1885.86

aSSDAEMAIFGEAAPYLR1869.87

aASPDAEMAAFGEAAPYLR1908.88

1596

02468

10

0 2 4 6 8 10 12 14Sample Number

Inte

nsi

ty

IVESMQR862.45

IVESLQSSLDAEIR1559.83

IVESMQSTLDAEIR1591.80

IVESMQR862.45

IVESLQSSLDAEIR1559.83

IVESMQSTLDAEIR1591.80

1283

01234567

0 2 4 6 8 10 12 14Sample Number

Inte

nsi

ty

LQTESGEYSR1169.54

LQTEAGEYSR1153.55

LQTETGEYSR1183.56

LQTESGEYSR1169.54

LQTEAGEYSR1153.55

LQTETGEYSR1183.56

1424

02468

101214

0 2 4 6 8 10 12 14Sample Number

Inte

nsi

ty LQNEVEDLMIDVER1702.83

LQNEVEDLMVDVER1688.82

LQNEVEDLMIDVER1702.83

LQNEVEDLMVDVER1688.82

1681

05

101520

0 2 4 6 8 10 12 14Sample Number

Inte

nsi

ty

ANLLQAE T EELR1386.72

ANLLQAE I EELR1398.76

ANLLQAE V EELR1384.74

ANLLQAE T EELR1386.72

ANLLQAE I EELR1398.76

ANLLQAE V EELR1384.74

1704

02468

10

0 2 4 6 8 10 12 14Sample Number

Inte

nsi

ty

VAEQEL M DA S ER1377.63

VAEQEL L DA S ER1359.68

VAEQEL L DA T ER1373.69

VAEQEL M DA S ER1377.63

VAEQEL L DA S ER1359.68

VAEQEL L DA T ER1373.69

Fig 2. Distribution of selected masses over the 14 samples

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.

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.

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

1820

1920

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.

MS spectrum of unseparated chicken dark meat digest

850 3200

1867 1876

Same spectrum, inset 1

Same spectrum, inset 2

Parent spectrum #6491869 peak is 5th highest

Parent spectrum #461 1869 peak is the highest

LC separation; TIC vs fraction #

MSMS spectrum of DLIELQALIDSHFEAR, Mascot score=95

y6b2

b4

HL

w,v9

y6

MSMS spectrum of aSSDAEMAIFGEAAPYLR, Mascot score=56

b4b5 b6

C.

150 1650

PSD-like msms spectrum of 1869 isotope cluster

y6

b2

b6

y6b5 b7y4

b8y3 b4

y14

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

y10

y12 y13b9b8y7ib6

PDGQVI

ib5PDGQV

ib4PDGQ

w13b2y3

A.w

d1

MYH peptidesw

d1

d2d3

wd1

wd2

d1d2

wd1d2

d2

w

d1

MYH peptides

PC1

PC

2

w

d1

d2d3

wd1

wd2

d1d2

wd1d2

w

d1

d2d3

wd1

wd2

d1d2

wd1d2

d2

PC

2

B.

w

d1

MYH peptidesw

d1

d2d3

wd1

wd2

d1d2

wd1d2

d2

w

d1

MYH peptides

PC

2

w

d1

d2d3

wd1

wd2

d1d2

wd1d2

w

d1

d2d3

wd1

wd2

d1d2

wd1d2

d2

B.