STJ detectors for protein detection

Physica C 468 (2008) 2009–2013

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Physica C journal homepage: www.elsevier.com/locate/physc

STJ detectors for protein detection Y. Kobayashi, M. Ukibe, K. Chiba-Kamoshida, H. Nakanishi, S. Shiki, K. Suzuki, M. Ohkubo * Research Institute of Instrumentation Frontier, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan

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Article history: Available online 3 June 2008 PACS: 07.20.Mc 82.80.Ms 87.14.E36.40.Qv Keywords: Superconducting tunnel junction Mass spectrometry Protein detection Fragmentation analysis MALDI

a b s t r a c t Time-of-flight mass spectrometry (TOF-MS) with matrix assisted laser desorption/ionization (MALDI) has become an essential spectrometry for a research field of bioscience. However, the mass limit of the conventional TOF-MS spectrometer frequently prevent us from measuring the mass values for large biomolecules without chemical or enzymatic digestion. Overcoming the circumstance, a TOFMS instrument with superconducting tunnel junction (STJ) detectors (Super TOF-MS) were developed. The direct observation of the kinetic energy for each ion by Super TOF-MS leads to the two considerable capabilities: the observation of large proteins up to 1 MDa and the ionic charge-state discrimination of them, which are impossible with conventional ion detectors. In this paper, it is demonstrated that the these advantages of Super TOF-MS is a powerful tool at performing a fragment analysis of, for example, immunoglobulin G (IgG) which is a large protein playing a key role in an immune system. Ó 2008 Elsevier B.V. All rights reserved.

1. Introduction Time-of-flight mass spectrometers (TOF-MS) equipped with superconducting detectors (Super TOF-MS) and matrix assisted laser deposition/ionization (MALDI) have a potential to detect biomolecules especially with high molecular weight (MW). The unit of Dalton is used in biosciences and equivalent to atomic mass unit. Compared with conventional molecule detectors of microchannel plates (MCPs) having a constraint of low sensitivity for large particles, the superconducting tunnel junction (STJ) detectors have high detection efficiency independent on MW. This advantage is realized by the fact that the STJ detectors measure not only the arrival times but also the kinetic energies of incoming particles [1], which are independent of MW when all ions are accelerated by a static voltage in TOF-MS. Actually, the practical high-mass limit of the STJ detectors has been reported with a large protein of immunoglobulin M (IgM) with a MW nearly 1 MDa [2], and polystyrene multimers up to 1 MDa [3]. In bioscience, MALDI TOF-MS is an essential method to identify peptide and protein molecules, which consist of sequences of amino acids. The difference between them is the numbers of amino acids: small numbers for peptides and more than 100 for pro-

* Corresponding author. Tel.: +81 29 861 5685; fax: +81 29 861 5730. E-mail address: [email protected] (M. Ohkubo). 0921-4534/$ - see front matter Ó 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.physc.2008.05.226

teins. Owing to the small number of the amino acids, the sequences of peptides can be determined by MALDI TOF-MS. This is one of the most important usages of MS. On the other hand, since most proteins have MWs more than 10 kDa, full sequence analysis without digestion is impossible. In this paper, we present fragment analysis for identifying the MWs of large fragments included as trace impurities in an IgG [4] solution by using the STJ detector. The word ‘‘fragment analysis” is different from the sequence analysis mentioned in the previous paragraph. This fragment analysis of large fragment molecules of IgG, which are much larger than small proteins, is realized with the high detection efficiency of the Super TOF-MS irrespective of MW. First, we evaluate practical mass resolution of the Super TOF-MS apparatus using different sizes of standard biomolecules to confirm whether it is enough to distinguish mass differences between fragments from IgG. In order to determine charge states of the fragments unambiguously, we utilized a scatter plots showing a relationship between kinetic energies versus TOF values for individual particle hit events. Because the kinetic energy is proportional to the charge-state of an ion, we can discriminate the ions with different charge numbers in the scatter plots: singly charged ions, doubly charged ions, and so on. It is demonstrated that the charge-state discrimination due to the kinetic energy measurement is extremely effective to simplify the complicated TOF-MS spectra to identify MWs of unexpected large fragments.

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2. Experimental 2.1. Biological samples

2.2. TOF-MS experiments with the STJ detector

130 110 90

FWHM = 5.67 [kDa]

70 50 30 10 138

An electron multiplier detector, which has an equivalent function of MCP and used in a conventional MALDI mass spectrometer, was replaced by a STJ detector fabricated at AIST. The details of the detector fabrication and design were reported in Ref. [5]. Briefly, the STJ detector has a size of 200  200 lm2 and a layer structure of Nb(100 nm)/Al(50 nm)/AlOx/Al(50 nm)/Nb(100 nm). The TOFMS experiments were performed in a linear mode with a N2 pulsed laser (337 nm). Ionized molecules are accelerated by a constant electrostatic voltage (V) of 17.5 kV. The kinetic energies of the ions are proportional to the charge number (z). This leads to an equation

m 2eV 2 / 2 t ; z L

150

Signal Intensity

Immunoglobulin G (IgG) from bovine serum (Sigma–Aldrich, MW ca. 150 kDa, purity: not less than 95% by SDS–PAGE) was dissolved in H2O/0.1% trifluoroacetic acid (TFA) as an IgG solution. Bovine serum albumin (BSA, 66.4 kDa) was purchased from Wako. NIST peptides, NIST-A of 1563 Da (Asp-Ala-Glu-Pro-AspIle-Leu-Glu-Leu-Ala-Thr-Gly-Tyr-Arg), NIST-B of 2950 Da (Lys-Ala-Gln-Tyr-Ala-Arg-Ser-Val-Leu-Leu-Glu-Lys-Asp-Ala-GluPro-Asp-Ile-Leu-Glu-Leu-Ala-Thr-Gly-Tyr-Arg), NIST-C of 1290 Da (Arg-Gln-Ala-Lys-Val-Leu-Leu-Tyr-Ser-Gly-Arg) were synthesized and purified at AIST. In the parentheses, the amino acid sequences are shown. Each peptide was dissolved in a 50% acetonitrile aqueous solution. Human angiotensin I of 1013 Da (Asp-Arg-Val-TyrIle-His-Pro-Phe-His-Leu) purchased from the PEPTIDE INSTITUTE Inc. was dissolved in distilled water at a concentration of 10 pmol/ll. As matrix solutions, sinapinic acid for IgG and BSA, and a-cyano-4-hydroxycinnamic acid (CHCA) for the NIST peptides and angiotensin were dissolved in an acetonitrile/0.1% TFA aqueous solution at the concentration of 10 mg/ml. The samples and the matrix solutions were mixed at a ratio of one-to-one and 1 ll of the mixed solutions were applied on stainless steal sample plates.

ð1Þ

where m, e, L, t are the MW, electron charge, flight distance, and TOF, respectively. The conventional TOF-MS instruments give only the m/z values obtained from Eq. (1). In addition to that, we can obtain the z values through the kinetic energy measurement with our TOF-MS apparatus. Therefore, the m values of individual molecules can be extracted.

3. Results and discussion In mass spectra, peak widths generally become wider with increasing MW mainly because of initial velocity distribution upon the ionization. The MW dependence of peak widths, which was obtained from the mass peaks of the NIST peptides, BSA, IgG, angiotensin, and their multimers, is shown in Fig. 1. The mass peak widths were estimated by a fitting with the Gaussian function, and the full width at half maximum (FWHM) values were evaluated. The MW dependence of the peak widths exhibits the performance of the Super TOF-MS instrument for the fragment analysis in this study. The scatter plot for the IgG measurement is shown in Fig. 2a. A mass spectrum of IgG and its multimers constructed from all events is shown in Fig. 2b. The mass spectra were constructed by

140

142

144

146

148

150

152

m/z [kDa] Fig. 1. The MW dependence of peak widths for the Super TOF-MS system. (a) FWHM values were estimated for standard peptides and proteins. The MW dependence of FWHM shows that the MWs for both a whole IgG molecule and fragments of that are enough distinguishable. (b) An example for Gaussian fitting on the mass peak for the intact IgG to obtain the corresponding FWHM value.

an accuracy of 500 Da. One can recognize the large peaks, which seem to be assigned to such IgG-related ions as singly charged monomers (145.5 kDa), singly charged dimmers (291.0 kDa), singly charged trimmers (430.0 kDa), doubly charged monomers (73.0 kDa), triply charged monomers (49.0 kDa), and triply charged dimmers (99.5 kDa). However, we cannot distinguish overlapped peaks of the ions that have the same m/z value, for example, the doubly charged dimmer and the singly charged monomer ions. The conventional MS instruments only produce the mass spectra shown in Fig. 2b. It should be noticed that many additional peaks appear in Fig. 2b besides all of the peaks expected from the intact IgG and its multimers. These unexpected peaks may be caused by fragments produced from the intact IgG by the pulse laser irradiation or some fragments existing as impurities in the IgG sample solution regardless of the high purity of the reagent. However, the conventional peak assignment using only m/z values is impossible because of the ambiguity over the z values. Thus, we used the z value discrimination by the kinetic energy measurement. In the scatter plot in Fig. 2a, we classified the events into three groups depending on the charge numbers from one to three. The mass spectra constructed for the singly charged ions, doubly charged ions, and triply charged ions separately are shown in Fig. 3a–c, which include the events inside the three rectangles in Fig. 2a, respectively. Because the peak patterns shown in Fig. 3 are remarkably simplified compared to that in Fig. 2b, one can more clearly distinguish the expected peaks for multimers and many unknown peaks with different charge states in Fig. 2b. Both of the singly charged and the doubly charged spectrum shown in Fig. 3a and b indicated

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Fig. 2. Super TOF-MS spectra. (a) Scatter plot (back) and intensity plot (front) of IgG. The horizontal and the vertical axes show the m/z value and the relative kinetic energy. In the intensity plot, a dark pixel represents that the high event numbers with the TOF and the energy. (b) A conventional mass spectrum drawn using events by all ions.

the existence of the fragments of the intact IgG, as will be discussed in the following paragraph. We summarize all the observed mass peaks in Tables 1 and 2. The m/z values correspond to the peaks observed in the singly, doubly and triply charged mass spectra in Fig. 3a–c. The m values are obtained by multiplying the m/z values and the respective charge numbers obtained from the kinetic energy measurement. In the fragment analysis, we assume that the IgG molecules are divided into two fragments, which are shown in the columns, Fragment A and Fragment B in Table 1. In the column, Fragment A, both of the observed fragment and IgG multimer peaks are listed. It is found that the m values for six out of the seven peaks of the singly charged ions well agree with those of the doubly charged ions. One peak with the smallest m value of 11.5 kDa in the singly charged mass spectrum exceptionally has no corresponding peak in the doubly charged spectrum, because of the high background signal in this mass region. The column, Fragment B, shows the peaks corresponding to the other part of the fragments to form the intact IgG molecule with the Fragment A. The column, A + B, shows the sum values of the MWs of the Fragments A and B, which should be comparable to the m value of the intact

IgG. One can notice the pairs of peaks, of which sum values are equal to the MW of the intact IgG, 145.5 kDa within the accuracy of the present experiment, for example, 11.5 kDa and 134.0 kDa, 23.0 kDa and 122.0 kDa. Consequently, it is shown that the observed small unknown peaks shown in Fig. 2b can be explained as fragment molecules assuming that the IgG molecule divides into two parts. It is well known that IgG is consists of twelve domains with almost the same MW. The most simplified fragmentation model of IgG is shown in Fig. 4. The model suggests that the peak of 11.5 kDa can be assigned to one of the twelve domains, and the rest part with the other eleven domains is detected as the peak of 134.0 kDa, which corresponds to Fig. 4a. The other fragments are also figured out by the fragmentation patterns in Fig. 4b–d. In Table 1, the expected number of the domains corresponding to each fragment is shown in the column, ‘‘domains”. Here, we should keep in mind that the fragmentation model in Fig. 4 is one candidate of the IgG fragmentations. Although the peak assignment shown in Table 1 is based on the assumption that the IgG molecule split in two fragments, there may be other fragmentations producing more than three fragments. Additional studies with the other biochemi-

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Signal Intensity (Ion Counts)

100

Table 1 Assignment of peaks appearing in spectra for singly, doubly, and triply charged ions

(c) Triply charged

90

Charge

80 70

Fragment A

49.0

60 Single (Fig. 3a)

50 40 30 20

73.5 98.0

145.5

10 0

0

50

100

150

200

250

300

350

400

450

m/z [kDa] Double (Fig. 3b)

Signal Intensity (Ion Counts)

400

(b) Doubly charged

73.0

350 300 250 200

49.0 62.0

150 100 50

Triple (Fig. 3c)

37.0 24.0

145.5

67.5 84.5 99.0

11.5

218.5

291.0

0 0

50

100

150

200

250

300

350

400

450

Signal Intensity (Ion Counts)

m/z [kDa] 500

350 300

(a)

145.5

450 11.5 400

Singly charged

23.0

250 200 150

73.5 47.5

100 50 0

0

50

134.0 122.0 99.5

100

150

168.5

200

289.0

250

300

432.5

350

400

450

m/z [kDa] Fig. 3. Charge discriminated Super TOF-MS spectra. (a) Singly, (b) doubly charged and (c) triply charged mass spectra.

cal analyses are required to specify the exact fragmentation patterns. It should be noted that the singly charged peak of 73.5 kDa can be assigned to the just half-cut fragment of IgG despite the fact that

Fragment B

m/z [kDa]

m [kDa]

Domains

m/z [kDa]

m [kDa]

Domains

11.5 23.0 47.5 73.5 99.5 122.0 134.0 145.5 289.0 432.5 11.5 24.0 37.0 49.0 62.0 67.5 73.0 145.5 218.5 291.0 49.0 98.0 145.5

11.5 23.0 47.5 73.5 99.5 122.0 134.0 145.5 289.0 432.5 23.0 48.0 74.0 98.0 124.0 135.0 146.0 291.0 437.0 582.0 147.0 294.0 436.5

1 134.0 2 122.0 4 99.5 6 73.5 8 47.5 10 23.0 11 11.5 Monomer – Dimmer – Trimmer – 2 62.0 4 49.0 6 37.0 8 24.0 10 11.5 11 – Monomer – Dimmer – Trimmer – Tetrammer – Monomer – Dimmer – Trimmer –

134.0 122.0 99.5 73.5 47.5 23.0 11.5 – – – 124.0 98.0 74.0 48.0 23.0 – – – – – – – –

11 10 8 6 4 2 1 – – – 10 8 6 4 2 – – – – – – – –

A+B m [kDa]

145.5 145.0 147.0 147.0 147.0 145.0 145.5 – – – 147.0 146.0 148.0 146.0 147.0 – – – – – – – –

The m values were obtained by multiplying the m/z values and the z values determined by the superconducting detector. We assume that IgG molecule splits into two parts and the observed mass peaks are listed in the columns, Fragment A and Fragment B. The intact IgG has twelve domains that have almost the same mass. See the text about more detailed explanation for the values in the table.

the cleavage of two disulfide bonds between the two heavy chains of IgG seems to be difficult. It is also interesting to see the interaction connecting the fragments and the intact IgG monomers, which appears as small peaks in Fig. 3a and b, and in Table 2. For example, the 168.5 kDa peak in Fig. 3c may be explained as a complex of the 23.0 kDa fragment and the 145.5 kDa IgG monomer. This suggests that the 23.0 kDa fragment strongly interacts with the intact IgG monomer, and thus the singly charged complex ions of 168.5 kDa are observed. Other peaks that may be assigned to the similar complex ions: 169.0 and 198.0 kDa as well as 168.5 kDa are shown in the column, Complex in Table 2. The peaks assigned to the complexes strongly support the existence of the fragment molecules of IgG. In the TOF-MS data, there is a possibility of the simultaneous multiple molecule hits. The double hit of the singly charged monomer might be observed as a doubly charged dimmer in the scatter plot. Furthermore, the post source decay (PSD) fragmentation of the doubly charged peak can produce an apparent peak in the singly charged mass spectrum. We always have to take into account such possibilities. In the present data, all the peaks of the doubly charged ions have the corresponding peaks in the singly charged

Table 2 Assignment of the complex peaks appearing in the mass spectra Charge

Single (Fig. 3a) Double (Fig. 3b) Triple (Fig. 3c)

Intact

Fragment

m/z [kDa]

m [kDa]

Domains

m/z [kDa]

m [kDa]

Domains

145.5 73.0 73.0 49.0

145.5 146.0 146.0 147.0

12 12 12 12

23.0 11.5 24.0 –

23.0 23.0 48.0 –

2 2 4 –

Intact + Fragment m [kDa]

Complex m [kDa]

168.5 169.0 194.0 –

168.5 169.0 198.0 220.5

We assume that the complex ions consist of an intact IgG monomer and a fragment molecule shown in Table 1. The m values for the intact IgG and the fragment molecules are listed in the columns, Intact and Fragment. The sum of the m values of both molecules are shown in the column, Intact + Fragment. The observed m/z values for the complexes are multiplied by the respective z values and listed in the column, Complex.

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Fig. 4. Example models of fragmentation patterns for IgG, (a) one domain separation, (b) two domain separation, (c) three domain separation, (d) separation into the halves by cleaving two disulfide bonds.

spectrum, and no typical PSD pattern is found in the scatter plot. Therefore, the contributions of the multiple hit events and the PSD may be negligibly small. 4. Conclusion We observed such large molecules as IgG monomers and multimers by the Super TOF-MS instrument. Utilizing the scatter plot representing the m/z values versus the kinetic energies, we can separately construct mass spectra of ions with the specific charge numbers. The charge discriminated mass spectra are significantly simple compared with the conventional mass spectra, which include the all charge states. It has been demonstrated that the z value discrimination is a powerful tool for the assignment of the unexpected mass peaks in complicated protein spectra.

Acknowledgements This project is granted by the Program of Japan Science and Technology Agency (JST-SENTAN). The authors express their thanks to T. Kinumi of AIST for sample preparation. References [1] M. Frank, S.E. Labov, G. Westmacott, H. Benner, Mass Spectrom. Rev. 18 (1999) 155. [2] R.J. Wenzel, U. Matter, L. Schultheis, R. Zenobi, Anal. Chem. 77 (2005) 4329. [3] M. Ohkubo, Y. Shigeri, T. Kinumi, N. Saito, M. Ukibe, Y.E. Chen, A. Kushino, A. Kurokawa, H. Sato, S. Ichimura, Nucl. Instrum. Methods A (2006) 779. [4] D. Voet, J.G. Voet, Biochemistry, John Wiley and Sons, Inc., London 1995. [5] Y. Chen, M. Ukibe, A. Kushino, M. Ohkubo, Nucl. Instrum. Methods A (2006) 4329.