Human Cardiac Troponin I is Post Translationally Modified by Arginine Methylation.

97

Figure 1: Immunoblots of 2.5 µg native full length human cardiac troponin I (cTnI).

A: Human cardiac troponin I identified as a methylated protein by anti-mono and dimethyl arginine antibody (α-ArgMe).  B:  Human cardiac troponin I recognised by anti-human cardiac troponin antibody (α-cTnI). Images are representative of two experimental repeats

MALDI/MS identified multi methylation peaks in the synthetic peptide of the inhibitory region of cTnI MALDI analysis of the peptide showed a peak at 1785.097 corresponding to the peptide and another peak corresponding to the sodium adduct. When this peptide was incubated with S-adenosyl methionine and protein arginine methyl transferase 1 (PRMT1) for 6 hours and a MALDI analysis done, more peaks were obtained at 1799.132 corresponding a methylated product of the peptide. Another peak (although weak) was observed at 1813.140 potentially corresponding to the dimethyl derivative of the peptide. These are in addition to other peaks at 1807.099 and 1821.111 potentially corresponding to the sodium adducts of the peptide and sodium adduct of the methylated peptide,  See figure 2.

Figure 2: MALDI-TOF mass spectrum of wild type peptide at 1785.09 Da  Upper panel: GKFKRPTLRRVRISA + SAM showing peaks of unmodified peptide at 1785.116 Da and peptide + sodium adducts at 1807.008 Da.

Lower panel: GKFKRPTLRRVRISA + SAM + PRMT1. Showing unmodified peptide peak at 1785.097 Da, monomethylated peptide peak at 1799.132, dimethylated peptide at 1813.140, peptide + sodium adduct peak at 1807.099 Da and sodium adduct + monomethylated peptide at 1821.111 Da. Images are representative of at least three experimental repeats.

MALDI/MS/MS identified the arginine methylated groups on the peptide, an MS/MS of the peptide was residue in the synthetic peptide of the inhibitory region conducted as a base line to study the fragmentation of the of cTnI ions in the methylated products, the mass of the observed

In order to identify the position of the addition of the methyl   ions are shown below, see figure 3, table 1.

Figure 3: MS/MS of m/z 1785.097 = GKFKRPTLRRVRISA. Fragmented unmodified peptide showing the b & y-ion series. Images are representative of at least three experimental repeats.

Table 1. Mass of fragmented GKFKRPTLRRVRISA peptides at m/z 1785.09

  a?

          b?         

Seq.

          y? 

 

1

      58.755

 G

 1785.103

15

2

185.910

K

14

3

      304.853     

332.826

F

1599.532

13

4

 K

 1452.409

12

5

      589.849

 616.848

R

 1324.219

11

6

 P

 1168.045

10

7

786.898

814.892

 T

9

8

      899.930     

927.952

L

969.950

8

9

    1083.994     

R

856.877

7

10

 1240.104

R

 700.834

6

11

    1311.248

        V

544.840

5

12

 R

 445.833

4

13

I

3

14

1695.821

S

176.880

2

15

A

1

Mass of peptides resulting from fragmentation of unmodified peptide with respect to a, b & y-ion series, Mascot score: 62; Expect: 9.7 E-06. Note only the mass of observed fragments is included. Empty cells mean that the corresponding ion was not observed.

When the peak at 1799.132 was fractionated into the component ions, it was possible to identify mono-methylation of arginine at position 10 (y6-ion) (GKFKRPTLRR*VRISA) in the MS/MS spectrum of peptide at m/z 1799.132, see figure 4, table 2. This arginine residue corresponds to R146 of cardiac troponin I protein. There was a +14 Da increase in mass of y6ion which was propagated through the rest of the spectrum, such that successive y-ions had +14 Da increases in the mass of fragments with respect to unmodified peptide in figure 3.

Figure 4: MS/MS of m/z 1799.11 = GKFKRPTLRRVRISA. Fragmented modified peptide showing the b & y-ion series. Images are representative of at least three experimental repeats

Onwuli, Donatus Onukwufor 

Table 2. Mass of fragmented GKFKRPTLRRVRISA peptides at m/z 1799.132 However,

a?

b?

Seq.

y?

1

             

 

G

1799.102 

 

15

2

176.898

K

 

1742.132

 

14

3

332.803

F

 

13

4

K

1466.482

12

5

 589.812

616.817

R

1338.165

11

6

P

1182.031

10

7

T

 9

8

 899.911

927.922

L

     983.291             

 8

9

1083.953

R

870.846

 7

10

R-

methyl

714.780

 6

11

 1325.138

V

544.832

 5

12

R

445.810

4

13

 

 I

289.800

3

14

1709.817

S

176.898

2

15

A

1

Fragmented modified peptide at 1799.132 Da showing a, b & y-ion series. Note +14 Da shift in y-ion series from y6, compared to unmodified peptide in table1. Mascot score: 42; Expect: 0.00095. Note only the mass of corresponding observed fragments is included. Empty cells mean that the corresponding ion was not observed. Highlighted in bold signify diagnostic ions.

Figure 5. MS/MS of m/z 1813.140 = GKFKRPTLRRVRISA. Fragmented modified peptide showing the b & y-ion series. Note the weak b11-H2O diagnostic ion. Images are representative of at least three experimental repeats

Table 3. Mass of fragmented GKFKRPTLRRVRISA peptides at m/z 1813.140

 

a? 

b? 

b?  –

H2O

Seq.

y? 

 

1

58.716

G

 

15

2

185.846

K

 

14

3

332.722

F

 

13

4

432.648

460.659

K

 

12

5

589.636

616.661

R

 

11

6

714.649

P

1197.63

10

7

786.625

814.627

797.48

T

9

8

899.652

927.649

910.56

L

   

8

9

1066.66

R

884.642

7

10

Rmethyl

729.270

6

11

 

1335.85  

V

5

12

Rmethyl

4

13

I

3

14

1696.097

1724.092

1706.08  

S

176.827

2

15

A

1

 Fragmented modified peptide at 1813.140 Da showing a, b & y-ion series. Mascot score: 19, Expect: 0.014. Only the mass of corresponding observed fragments is  included. Empty cells mean that the corresponding ion was not observed. Highlighted  in bold signify diagnostic ions.

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