intact protein analysis by maldi tandem time-of … protein analysis by maldi tandem time-of-flight...

Intact Protein Analysis by MALDI Tandem Time-of-flight Mass Spectrometry

Melanie Lin1, Jennifer M. Campbell1, Dieter R. Mueller2, Urs Wirth2

1Applied Biosystems, Framingham, MA01701, USA 2Functional Genomics, Novartis Pharma AG, CH-4002 Basel, Switzerland

ABSTRACT

Direct MS/MS analysis of small, singly charged protein ions by tandem TOFMS is demonstrated

for proteins up to a molecular mass of 12 kDa. The MALDI generated singly charged precursor

ions form predominantly product ions resulting from metastable fragmentation at aspartyl and

prolyl residues. Additional series of C-terminal sequence ions provide in some cases sufficient

information for protein identification. The amount of sample required to obtain good quality

spectra is in the high femtomolar to low picomolar range. Within this range, MALDI-MS/MS

using TOF/TOFTM ion optics provides now the opportunity for direct protein identification and

partial characterization without prior enzymatic hydrolysis.

INTRODUCTION

Tandem mass spectrometry has been widely adopted as a means to generate complete or partial

sequence information of peptides, and, thereby, an efficient way to identify the corresponding proteins

via database searching. For the MS/MS analysis of large, and predominantly singly charged, peptide

and intact protein ions that are formed in the MALDI process, there are, however, limitations in the use

of many of the existing technologies. If quality MS/MS spectra could be routinely recorded from higher

mass ions, it would not only be possible to use more rapid protein hydrolyses (such as cyanogen

bromide) that form higher mass (i.e., > 4000 Da) peptides, but it would also enable the direct

identification of smaller proteins (e.g., biomarkers) without resorting to enzymatic digestion.

Although initially designed to rapidly identify a large number of proteins based on MS/MS analyses of

tryptic peptides1, this study demonstrates that the MALDI tandem time of flight mass spectrometer

using TOF/TOFTM ion optics also provides a powerful platform for MS/MS of smaller protein and large

peptide ions. In this report, we will demonstrate MS/MS analysis of selected small proteins using

Applied Biosystems 4700 Proteomics Analyzer with TOF/TOFTM ion optics.

MATERIALS AND METHODS

Sample Preparation

The proteins such as Insulin, Thioredoxin and r-Hirudin were dissolved in 50% ACN, 0.3% TFA

solution and further diluted by alpha-cyano-4-hydroxycinnamic acid matrix solution (5 mg/mL in 50%

ACN, 0.3% TFA) to concentrations of 10 pmol, 1 pmol and 200 fmol/ l, respectively. 1 l of each

protein in matrix solution was spotted onto the MALDI plate.

Mass Spectrometry

Samples were analyzed using the Applied Biosystems 4700 Proteomics Analyzer with TOF/TOFTM ion

optics. Both MS and MS/MS data were acquired with a Nd:YAG laser with 200 Hz repetition rate and

up to 4000 shots were accumulated for each spectrum. MS/MS mode was operated with 2 keV

collision energy and air was added as the collision gas such that nominally “single collision conditions”

were achieved. Both MS and MS/MS data were acquired using the instrument default calibration,

without applying internal or external calibration. Sequence tag searches were performed with the

program Peptide Search (MDS Proteomics, Denmark).

of 4 ASMS 2003 – Poster Number T-239: Lin, Campbell, Mueller, Wirth

Poster Number T-239

RESULTS

Figure 3. Partial MS/MS spectrum of [M+H]+ of Insulin

(m/z 5730; 1 pmol/ L). Sequence tag in the low mass

region.

Figure 4. Partial MS/MS spectrum of [M+H]+ of r-Hirudin

(m/z 6964.5; 1 pmol / L). Major fragments primarily at

prolyl and aspartyl residues.


(m/z 5730; 1 pmol/ L), predominant isoforms indicated.

Resolution of the product ions in the mass range shown

are on average 6500 FWHM.


(m/z 5730; 1 pmol/ L ). It indicates one cleaved SS-

bridges in the fragment y17b.

3282.0 3327.4 3372.8 3418.2 3463.6 3509.0

2244.0

0

10

20

30

40

50

60

70

80

90

100

[B-Chain(-SSH)2+H]+

m/z

[B-Chain(-SH/-SSH)+H]+

[B-Chain(-SH/-S)]+

[B-Chain(-SH/-SH)-H2S +H]+

[B-Chain(-SH/-SH)-2H2S +H]+

3330.49

3430.35

3396.45

3364.40

3462.40

30

100 3431.54

3430.35

3429.51

3428.33

3432

3282.0 3327.4 3372.8 3418.2 3463.6 3509.0

2244.0

0

10

20

30

40

50

60

70

80

90

100

[B-Chain(-SSH)2+H]+

m/z

[B-Chain(-SH/-SSH)+H]+

[B-Chain(-SH/-S)]+

[B-Chain(-SH/-SH)-H2S +H]+

[B-Chain(-SH/-SH)-2H2S +H]+

3330.49

3430.35

3396.45

3364.40

3462.40

30

100 3431.54

3430.35

3429.51

3428.33

3432

3441.0 4170.6 4900.2 5629.8 6359.4 7089.0

Mass (m/z)

1398.0

0

10

20

30

40

50

60

70

80

90

100

% I

nte

ns

ity

4700 MS/MS Precursor 6964 Spec #1=>AdvBC(32,0.5,0.1)=>NF1.0[BP = 30.1, 1

-D

y60

-D

b55-P

b47

-D

b53

[M+H]+

-E

b43

6385.67

5667.21

5495.16

4433.28

6964.71

4816.63

m/z

3441.0 4170.6 4900.2 5629.8 6359.4 7089.0

Mass (m/z)

1398.0

0

10

20

30

40

50

60

70

80

90

100

% I

nte

ns

ity


-D

y60

-D

b55-P

b47

-D

b53

[M+H]+

-E

b43

6385.67

5667.21

5495.16

4433.28

6964.71

4816.63

m/z

-D

y60

-D

b55-P

b47

-D

b53

[M+H]+

-E

b43

6385.67

5667.21

5495.16

4433.28

6964.71

4816.63

m/z

In the product ion spectrum of [M+H]+ (m/z 5730.6) of Insulin (Figure 1), all fragment ions show good

resolution (on average 6500, FWHM; cf. also inset in Figure 1). The quality of the data allows

assignment of the predominant isoforms to the individual isotope clusters of the B-chain fragments.

Accuracy of mass assignment is 60 ppm in mean mass deviation. The triplet around m/z 1934 (Figure

2) indicates one symmetrically and asymmetrically cleaved SS-bridge, now contained in the fragment

ion y17b. Partial sequence information is obtained from the B-chain in the low mass region (Figure 3).

Sequence tag search detects insulin B-chain as outstanding hit. The MS/MS spectrum of r-Hirudin is

dominated by C-terminal cleavage at aspartic acid residues (Figure 4). It is also of note that relatively

large partial sequence stretches, not related to acid- or proline-directed fragmentation were observed in

the MS/MS spectrum of [M+H]+ of intact r-Hirudin in the low mass region (Figure 5). The amino acid

coverage from this region is specific enough for protein identification through a BLAST search.

1807.0 1854.6 1902.2 1949.8 1997.4 2045.0

Mass (m/z)

268.7

0

10

20

30

40

50

60

70

80

90

100

% In

ten

sit

y

4700 MS/MS Precursor 5734 Spec #1=>SM23[BP = 43.1, 6410]

[y17b(-SSH)][y17b(-SH)]

[y17b(-SH)-H2S]

1900.82

1934.81

1966.73

m/z

1807.0 1854.6 1902.2 1949.8 1997.4 2045.0

Mass (m/z)

268.7

0

10

20

30

40

50

60

70

80

90

100

% In

ten

sit

y

4700 MS/MS Precursor 5734 Spec #1=>SM23[BP = 43.1, 6410]

[y17b(-SSH)][y17b(-SH)]

[y17b(-SH)-H2S]

1900.82

1934.81

1966.73

m/z

895.0 1023.2 1151.4 1279.6 1407.8 1536.0

Mass (m/z)

1890.8

0

10

20

30

40

50

60

70

80

90

100

% I

nte

nsit

y

4700 MS/MS Precursor 5734 Spec #1=>SM11=>AdvBC(11,0.5,0.1)[BP = 43.1, 9968]y9b

y10b

y11b

1272.86

1215.66

1086.59

895.0 1023.2 1151.4 1279.6 1407.8 1536.0

Mass (m/z)

1890.8

0

10

20

30

40

50

60

70

80

90

100

% I

nte

nsit

y

4700 MS/MS Precursor 5734 Spec #1=>SM11=>AdvBC(11,0.5,0.1)[BP = 43.1, 9968]y9b

y10b

y11b

1272.86

1215.66

1086.59

y9b

y10b

y11b

1272.86

1215.66

1086.59

y10b

y11b

1272.86

1215.66

1086.59

m/z


Poster Number T-239

Figure 5. Partial MS/MS spectrum of [M+H]+ of r-Hirudin

(m/z 6964.5; 1 pmol / L). Sequence stretch provides

enough information for a correct protein identification.

Figure 6. MS/MS spectrum of [M+H]+ of Thioredoxin

(m/z 11674.4; 10 pmol/ L). Major fragments primarily at

prolyl and aspartyl residues.

Figure 7. MS/MS spectrum of

[M+H]+ of Thioredoxin (m/z

11674.4; 10 pmol/ L).

Nonspecific y ions are

observed.

47]47]

m/z

1966.0 2208.8 2451.6 2694.4 2937.2 3180.0

Mass (m/z)

489.

0

10

20

30

40

50

60

70

80

90

100

% I

nte

ns

ity

4700 MS/MS Precursor 6964 Spec #1=>NF1.0=>BC[BP = 30.1, y20

y22

y25

y26

y18

2147.6

2372.5

2473.4

y21

2817.9

2716.8

2530.8

y24

2660.6

y23

2916.9

y19

2276.0

m/z

1966.0 2208.8 2451.6 2694.4 2937.2 3180.0

Mass (m/z)

489.

0

10

20

30

40

50

60

70

80

90

100

% I

nte

ns

ity

4700 MS/MS Precursor 6964 Spec #1=>NF1.0=>BC[BP = 30.1, y20

y22

y25

y26

y18

2147.6

2372.5

2473.4

y21

2817.9

2716.8

2530.8

y24

2660.6

y23

2916.9

y19

2276.0

3801.0 5431.6 7062.2 8692.8 10323.4 11954.0

Mass (m/z)

1504.

0

10

20

30

40

50

60

70

80

90

100

% I

nte

nsit

y


-D

y93

m/z

-P

y45-D

y88-D

y82

-D

y65

-D

y61

-D

y47

-D

y98

-D

y95

-D

y99

[M+H]+

8875.09

10536.00

10185.81

9969.64

9443.636914.65

6486.26

4912.53

4670.3210649.89

11673.99

3801.0 5431.6 7062.2 8692.8 10323.4 11954.0

Mass (m/z)

1504.

0

10

20

30

40

50

60

70

80

90

100

% I

nte

nsit

y


-D

y93

m/z

-P

y45-D

y88-D

y82

-D

y65

-D

y61

-D

y47

-D

y98

-D

y95

-D

y99

[M+H]+

8875.09

10536.00

10185.81

9969.64

9443.636914.65

6486.26

4912.53

4670.3210649.89

11673.99

3717.0 3896.8 4076.6 4256.4 4436.2 4616.0

Mass (m/z)

55.3

0

10

20

30

40

50

60

70

80

90

100

% In

ten

sit

y

4700 MS/MS Precursor 11673 Spec #1=>AdvBC(32,0.5,0.1)=>NF1.0=>SM29

y36

y38

y37

y39

y41y42

Y43-H2O

3717.0 3896.8 4076.6 4256.4 4436.2 4616.0

Mass (m/z)

55.3

0

10

20

30

40

50

60

70

80

90

100

% In

ten

sit

y


y36

y38

y37

y39

y41y42

Y43-H2O

y36

y38

y37

y39

y41y42

Y43-H2O

4591.0 4754.6 4918.2 5081.8 5245.4 5409.0

Mass (m/z)

901.3

0

10

20

30

40

50

60

70

80

90

100

% In

ten

sit

y

4700 MS/MS Precursor 11673 Spec #1=>AdvBC(32,0.5,0.1)=>NF1.0=>SM11[B

-P

y45

-D

y47

y48 y49 y50 y51y46

4591.0 4754.6 4918.2 5081.8 5245.4 5409.0

Mass (m/z)

901.3

0

10

20

30

40

50

60

70

80

90

100

% In

ten

sit

y


-P

y45

-D

y47

y48 y49 y50 y51y46

5465.0 5647.8 5830.6 6013.4 6196.2 6379.0

Mass (m/z)

52.5

0

10

20

30

40

50

60

70

80

90

100

% In

ten

sit

y


y53y54

y59

y58

y57y56

y60

5465.0 5647.8 5830.6 6013.4 6196.2 6379.0

Mass (m/z)

52.5

0

10

20

30

40

50

60

70

80

90

100

% In

ten

sit

y


y53y54

y59

y58

y57y56

y60

6408.0 6519.8 6631.6 6743.4 6855.2 6967.0

Mass (m/z)

282.5

0

10

20

30

40

50

60

70

80

90

100

% I

nte

ns

ity

4700 MS/MS Precursor 11673 Spec #1=>AdvBC(32,0.5,0.1)=>NF1.0=>SM11[BP -D

y61

-D

y65

y63

y64

y62

6408.0 6519.8 6631.6 6743.4 6855.2 6967.0

Mass (m/z)

282.5

0

10

20

30

40

50

60

70

80

90

100

% I

nte

ns

ity


y61

-D

y65

y63

y64

y62

a. c.

b.d.

3784.46

3897.55

3954.93

4118.71

4345.024415.25

4498.03

5566.585666.26

5878.596008.50

6066.42

6194.84

6356.69

4670.32

4783.02

4912.53

5026.31 5140.54 5256.71 5367.39

6486.26

6602.10 6672.70

6685.53

6914.65

m/z

m/z m/z

m/z3717.0 3896.8 4076.6 4256.4 4436.2 4616.0

Mass (m/z)

55.3

0

10

20

30

40

50

60

70

80

90

100

% In

ten

sit

y


y36

y38

y37

y39

y41y42

Y43-H2O

3717.0 3896.8 4076.6 4256.4 4436.2 4616.0

Mass (m/z)

55.3

0

10

20

30

40

50

60

70

80

90

100

% In

ten

sit

y


y36

y38

y37

y39

y41y42

Y43-H2O

y36

y38

y37

y39

y41y42

Y43-H2O

4591.0 4754.6 4918.2 5081.8 5245.4 5409.0

Mass (m/z)

901.3

0

10

20

30

40

50

60

70

80

90

100

% In

ten

sit

y

4700 MS/MS Precursor 11673 Spec #1=>AdvBC(32,0.5,0.1)=>NF1.0=>SM11[

-P

y45

-D

y47

y48 y49 y50 y51y46

4591.0 4754.6 4918.2 5081.8 5245.4 5409.0

Mass (m/z)

901.3

0

10

20

30

40

50

60

70

80

90

100

% In

ten

sit

y

4700 MS/MS Precursor 11673 Spec #1=>AdvBC(32,0.5,0.1)=>NF1.0=>SM11[

-P

y45

-D

y47

y48 y49 y50 y51y46

5465.0 5647.8 5830.6 6013.4 6196.2 6379.0

Mass (m/z)

52.5

0

10

20

30

40

50

60

70

80

90

100

% In

ten

sit

y


y53y54

y59

y58

y57y56

y60

5465.0 5647.8 5830.6 6013.4 6196.2 6379.0

Mass (m/z)

52.5

0

10

20

30

40

50

60

70

80

90

100

% In

ten

sit

y


y53y54

y59

y58

y57y56

y60

6408.0 6519.8 6631.6 6743.4 6855.2 6967.0

Mass (m/z)

282.5

0

10

20

30

40

50

60

70

80

90

100

% I

nte

ns

ity


y61

-D

y65

y63

y64

y62

6408.0 6519.8 6631.6 6743.4 6855.2 6967.0

Mass (m/z)

282.5

0

10

20

30

40

50

60

70

80

90

100

% I

nte

ns

ity


y61

-D

y65

y63

y64

y62

a. c.

b.d.

3784.46

3897.55

3954.93

4118.71

4345.024415.25

4498.03

5566.585666.26

5878.596008.50

6066.42

6194.84

6356.69

4670.32

4783.02

4912.53

5026.31 5140.54 5256.71 5367.39

6486.26

6602.10 6672.70

6685.53

6914.65

m/z

m/z m/z

m/z

Examples of MS/MS analysis on Thioredoxin (m/z 11674.4, [M+H]+) demonstrate the high-mass

capability of the TOF/TOFTM ion optics. As discussed above, the major fragments are again primarily

formed at aspartic acid residues (Figure 6) and the dominant product ions observed are identical to

those previously described in earlier work using PSD analysis.2 It is also interesting to note that

extended y-ion series are observed in proximity to the Asp-cleavages (Figure 7a-d). This partial

sequence information can again be transformed into sequence tags for protein identification. Figures

8 and 9 show MS/MS analyses of Thioredoxin at 1 pmol and 200 fmol respectively, demonstrating the

sensitivity of the TOF/TOF instrument in this mass range.


Poster Number T-239


(m/z 11674.4; 200 fmol/ L).

2684.0 4567.4 6450.8 8334.2 10217.6 12101.0

Mass (m/z)

544.0

0

10

20

30

40

50

60

70

80

90

100

% In

ten

sit

y

4700 MS/MS Precursor 11673 Spec #1=>NF1.0=>AdvBC(32,0.5,0.1)[BP = 70.1

m/z

-P

y45

-D

y65

-D

y61

-D

y47

-D

y93

-D

y82

-D

y88

-D

y95

-D

y98

-D

y99

[M+H]+

10651.9610536.89

9972.17

9443.36

8874.14

6914.84

6486.35

4912.57

4670.09

11673.56

10188.25

2684.0 4567.4 6450.8 8334.2 10217.6 12101.0

Mass (m/z)

544.0

0

10

20

30

40

50

60

70

80

90

100

% In

ten

sit

y

4700 MS/MS Precursor 11673 Spec #1=>NF1.0=>AdvBC(32,0.5,0.1)[BP = 70.1

m/z

-P

y45

-D

y65

-D

y61

-D

y47

-D

y93

-D

y82

-D

y88

-D

y95

-D

y98

-D

y99

[M+H]+

10651.9610536.89

9972.17

9443.36

8874.14

6914.84

6486.35

4912.57

4670.09

11673.56

10188.25


(m/z 11674.4; 1 pmol/ L).

3200.0 4981.4 6762.8 8544.2 10325.6 12107.

Mass (m/z)

1.1

0

10

20

30

40

50

60

70

80

90

100

% In

ten

sit

y

4700 MS/MS Precursor 11673 Spec #1=>SM25=>SM45[BP = 4910.7, 1]

6484.13

-P

y45

-D

y65

-D

y88

-D

y61

-D

y47

-D

y93

-D

y99

-D

y95

[M+H]+

11670.10

10653.02

10535.00

10184.00

9428.526913.94

4910.25

4670.47

m/z

3200.0 4981.4 6762.8 8544.2 10325.6 12107.

Mass (m/z)

1.1

0

10

20

30

40

50

60

70

80

90

100

% In

ten

sit

y

4700 MS/MS Precursor 11673 Spec #1=>SM25=>SM45[BP = 4910.7, 1]

6484.13

-P

y45

-D

y65

-D

y88

-D

y61

-D

y47

-D

y93

-D

y99

-D

y95

[M+H]+

11670.10

10653.02

10535.00

10184.00

9428.526913.94

4910.25

4670.47

m/z

6484.13

-P

y45

-D

y65

-D

y88

-D

y61

-D

y47

-D

y93

-D

y99

-D

y95

[M+H]+

11670.10

10653.02

10535.00

10184.00

9428.526913.94

4910.25

4670.47

m/z

CONCLUSIONS

Intact proteins up to 12,000 Da were analyzed in the range from 200 fmol to 10 pmol by MALDI

tandem mass spectrometry with TOF/TOFTM ion optics. MS/MS spectra of singly-charged precursor

ions of these proteins show predominantly product ions resulting from fragmentation at aspartyl and

prolyl residues. The additional ion series observed in selected mass regions can provide sequence

tags for positive protein identification. Metastable decomposition combined with high energy CID of

intact small proteins provides now the opportunity for direct protein identification via database

searches or for direct site-mapping of post-translational modifications without having to resort to

enzymatic digests.

Reference

1. Vestal M., Campbell J. Tandem Time-of-Flight Mass Spectrometry. Methods in Enzymology In

press, 2003.

2. Yu W, Vath JE, Huberty MC, Martin SA. Anal. Chem. 1993; 65: 3015-3023.


Poster Number T-239

intact protein analysis by maldi tandem time-of … protein analysis by maldi tandem time-of-flight...

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