Journal of The American Society for Mass Spectrometry

, Volume 25, Issue 3, pp 303–309

Advantages of Monodisperse and Chemically Robust “SpheriCal” Polyester Dendrimers as a “Universal” MS Calibrant

Authors

    • Department of ChemistryTulane University
  • Brittany K. Myers
    • Department of ChemistryTulane University
  • Jonas Bengtsson
    • Polymer Factory Sweden AB
  • Michael Malkoch
    • Polymer Factory Sweden AB
    • Department of Fiber and Polymer Technology, School of Chemical Science and EngineeringKTH Royal Institute of Technology
Research Article

DOI: 10.1007/s13361-013-0777-8

Cite this article as:
Grayson, S.M., Myers, B.K., Bengtsson, J. et al. J. Am. Soc. Mass Spectrom. (2014) 25: 303. doi:10.1007/s13361-013-0777-8

Abstract

The utilization of dendrimer calibrants as an alternative to peptides and proteins for high mass calibration is explored. These synthetic macromolecules exhibited a number of attractive advantages, including exceptional shelf-lives, broad compatibility with a wide range of matrices and solvents, and evenly spaced calibration masses across the mass range examined, 700–30,000 u. The exceptional purity of these dendrimers and the technical simplicity of this calibration platform validate their broad relevance for high molecular weight mass spectrometry.

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Figure

Key words

CalibrantDendrimerCalibrationPolymerMALDI-TOF MS

1 Introduction

Mass spectrometry has become an invaluable technique for the characterization of both biological [1, 2] and synthetic macromolecules [35] because of the abundance of information that can be obtained about macromolecular structure and molecular weight distributions from their molecular ions. For example, MALDI-TOF MS analysis is perhaps the most sensitive and accurate method for determining the identity of end groups on synthetic polymers as well as elucidating the overall molecular weight distribution. Likewise, MALDI-TOF MS, ESI, and tandem MS techniques provide arguably the most simple and rapid routes for determining the primary structure of peptides and proteins, as well as the extent of post-translational modifications. Advances in MS instrument design have yielded substantial improvements in resolving power, mass accuracy, and detection limits, particularly for higher molecular weight macromolecules; however, because the observed mass scale can shift with changes in acquisition parameters, regular calibration is critical for providing highly accurate molecular weight calculations. Therefore, a critical requirement for the continued advancement of high molecular weight MS is the development of high mass calibration platforms that are rapid, technically simple, and cost efficient, yet consist of robust species that exhibit extended shelf-lives at ambient conditions, and sufficient versatility for both internal and external calibration with a broad range of analytes.

Traditionally, peptides and proteins have been used as high molecular weight calibrants because of their monodispersity and availability; however, their cost and stability can vary drastically. Although many can be harvested from biological sources, their purification can be demanding, and synthetic routes to peptides are expensive because of the demanding serial nature of their synthesis. Regarding stability, some peptides exhibit long-term stability at room temperature, but many are susceptible to chemical degradation via deamination or hydrolysis as well as the action of ubiquitous proteases. Other calibration systems have seen regular use, including inorganic ion clusters and polydisperse polymers. While these alternatives are usually orders of magnitude less expensive and exhibit longer shelf-lives, they are most useful for low mass calibrations (>10,000). Their use for calibration at higher masses can be complicated by low signal or uncertain signal assignments resulting from their mass dispersity. Dendrimers exhibit the potential to combine the advantages of the approaches above: they can be efficiently prepared as monodisperse compounds, in high yields, and with vastly improved stability relative to traditional peptide or protein calibrants. Below, we confirm the monodispersity, broad matrix and solvent compatibility, and exceptional stability of “SpheriCal,” a polyester dendrimer-based MS calibration system.

2 Experimental

2.1 Materials

All matrices, sodium trifluoroacetate, and potassium trifluoroacetate were purchased from Sigma-Aldrich (St. Louis, MO, USA), and used without further purification. Steel-target plates (MTB 384) were purchased from Bruker (Billerica, MA, USA). SpheriCal-Neat standards were obtained from Polymer Factory Sweden AB (Stockholm, Sweden). The protein calibrant “Protein Standard II” was purchased from the online Bruker Care Shop. ACTH fragment (18–39) was purchased from Sigma-Aldrich and insulin was purchased from Protea Biosciences (Morgantown, WV, USA).

2.2 Mass Spectrometry: Sample Preparation and Data Analysis

MALDI-TOF MS data were acquired on two different instruments. The first was a Bruker Autoflex III (Bruker Daltonics, Billerica, MA, USA) in positive ion mode using the flexControl 3.0 software. Reflector mode was utilized for samples below 7.5 kDa, and linear mode for samples above 7.5 kDa. Samples were deposited on the target plate via the dried droplet method by mixing 10 μL of 10 mg/mL of matrix, 1 μL of 2 mg/mL of sodium trifluoroacetate, and 2 μL of 2 mg/mL of dendritic calibrant. Representative acquisition parameters were: ion source 1, 19.0 kV; ion source 2, 16.3 kV; lens, 9.1 V, reflector; pulse ion extraction, 40 ns; detector, 1.905 kV. Approximately 1000 laser shot/spectra were obtained with a 1 kHz smartbeam II Nd:YAG laser and the laser intensity adjusted according to the signal intensity. All spectra reported were obtained using the Bruker Autoflex III mass spectrometer, unless otherwise noted. The second instrument used was a Bruker UltraFlex MALDI-TOF MS with a SCOUT-MTP ion source (Bruker Daltonics) equipped with a N2-laser (337 nm), a gridless ion source, and a reflector. Samples were prepared using 10 μL of a 10 mg/mL matrix solution of 9-nitroantracene in THF, 5 μL of a 1 mg/mL solution of the counter-ion source NaTFA, and 5 μL of a 1 mg/mL solution of analyte and deposited on the target plate via the dried droplet method. 9-Nitroanthracene and NaTFA dissolved in THF were used for sample preparation. All spectra were acquired using a reflector-positive method with an acceleration voltage of 25 kV and a reflector voltage of 26.3 kV. The laser was set to the lowest value possible to acquire high resolution spectra. The instrument was calibrated using SpheriCal calibrants purchased from Polymer Factory Sweden AB. The protein calibrant used for comparison was prepared following the instructions provided. The obtained spectra were analyzed with FlexAnalysis Bruker Daltonics ver. 2.2. The mass scale was preliminarily calibrated externally against SpheriCal dendritic calibrants obtained from Polymer Factory Sweden AB. Data analysis was carried out using FlexAnalysis Bruker Daltonics ver. 2.2 or 3.0 software, but the reported spectra are shown without smoothing, baseline subtraction, or further data modifications. Protein shelf life studies were carried out at room temperature and spectra obtained using the same proportions of analyte to a-cyano-4-hydroxycinnamic acid (CHCA) as a matrix. Internal calibration with insulin was carried out by combining 10 μL of 10 mg/mL of CHCA matrix, 1 μL of 2 mg/mL of sodium trifluoroacetate (omitted for spectrum generated without sodium), 2 μL of 2 mg/mL of dendritic calibrant, and 2 μL of 2 mg/mL ACTH (fragment 18–39). Once mixed, 3 μL of this solution was deposited on the target plate.

3 Results and Discussion

3.1 Synthesis

The dendrimers described in this study were synthesized using a divergent dendronization process initiated from one of four different core molecules (A, B, C, or D) and an acid anhydride monomer based on bis(hydroxymethyl)propanoic acid or “bis-MPA” using previously reported techniques [6]. Each dendronization was continued through five synthetic iterations (or generations), yielding a total of 20 unique compounds. While some of the earliest reported dendrimers using other chemistries exhibited structural impurities that result from well-known side reactions during their synthesis [7], this particular bis-MPA-based synthesis exhibits exceptionally monodisperse molecules (Figures 1, 2, 3). The observed monodispersity is a result of the combination of highly efficient esterification and deprotection reactions that can be easily driven to completion, as well as the lack of detectable side reactions [8]. In addition, the robust nature of the pivalate ester gives these compounds hydrolytic stability beyond what is usually associated with esters, particularly under acidic conditions (e.g., pH 4.5) [9], making them appropriate for internal calibration with most peptide and protein analytes.
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Figure 1

Representative structure of the SpheriCal calibrant, PFS5-A (above), with an average molecular weight [M + Na]+ of 15,171.2. Mass spectra (below) obtained using a Bruker Ultraflex MALDI-TOF mass spectrometer

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Figure 2

Comparison of the observed spectra for protein and SpheriCal calibrants (above): (a) commercial protein standard containing [trypsinogen + H]+ in linear mode, (b) PFS5 in linear mode and (c) PFS5 in reflector mode. The resolution calculated for a representative peak in each spectrum is labeled in red, and expanded spectra are provided in Supplementary Figure S1. Data were obtained using Bruker Ultraflex MALDI-TOF mass spectrometer

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Figure 3

Mass spectra for each of the five calibration sets providing a total of 20 evenly spaced calibration points from m/z 700–30,000. Observed ions are sodium adducts, [M + 22.98]+ with exact monoisotopic masses reported for PFS1-3 and average masses reported for PFS4-5

3.2 Molecular Weight Range and Spacing

An ideal synthetic calibrant system should exhibit a set of known, well-defined, and well-spaced m/z signals across a wide mass range. While synthetic polymers [10, 11], ion clusters [1216], resorcinarene clusters [17], and oligosaccharides [18] provide an efficient solution for lower molecular weight calibration (e.g., below 5000 u), the relatively narrow spacing of oligomers (e.g., 44.03 for PEG, m/z = 104.06 for polystyrene (PS), 162.05 for oligosaccharides of hexose, and 259.81 for CsI) and uncertainty in end groups, cations, or trace repeat unit impurities can result in vague peak assignments for higher mass ranges. For mass ranges above 10,000, many of these calibration platforms provide negligible signal. One exception, CsI cluster ions, have demonstrated signal as high as 20,000 u for MALDI-TOF MS calibration in linear mode; however, when the higher-resolution reflector mode was attempted, CsI cluster were only observed up to 3500 u [19]. While synthetic polymers are widely available even in high mass ranges, the relatively narrow peak spacing can obscure accurate identification. Some proteins are also available for higher molecular weights, but there are large gaps in the mass ranges for which proteins are readily available, many proteins suffer from inherent instability, their observed signals can broaden substantially because of multiple modes of ionization, and there can be uncertainty regarding their exact molecular weight, especially for larger proteins [20]. SpheriCal dendritic calibrants (Figure 1) offer unique advantages for high molecular weight MS calibration because of their exceptional purity and synthetic versatility, yielding well-spaced, well-resolved calibrant signals between m/z 700 and 30,000 (see Table 1). In addition, because the described structures exhibit multiple ester groups but lack amino and carboxylate functionalities, they exhibit a relatively narrow mass signal corresponding to ionization via adduct formation with a single cation (in this study, Na+ provided by sodium trifluoroacetate). This improvement in resolution is visible during the comparison of protein calibrants and SpheriCal (Figure 2). While it was difficult to achieve useful signal for [trypsinogen + H]+ at m/z 23982 in reflector mode, linear mode yielded sufficient signal for calibration with a resolution of 212 (Figure 2a and Supplementary Figure S1a). However, the SpheriCal calibrant, PFS5, exhibits four strong signals in the same mass range in both linear and reflector modes (Figure 2b, c and Supplementary Figure S1b, c), and demonstrates a substantial improvement in resolution (as high as 2500).
Table 1

Names, Formulas, and Exact Masses of the 20 Dendrimers Evaluated in this Study

SpheriCal dendritic calibrant

Single PEAK codes

Formula

Exact mass (m/z)

Resolution (Auto-flex III)

Resolution (Ultra-flex)

Peptide low range SET 1

PFS1-A

C41H48O12

732.3146

8297

9339

PFS1-B

C53H60O16

952.3881

8707

10815

PFS1-C

C65H72O20

1172.462

10045

11974

PFS1-D

C82H94O25

1478.608

12410

13720

Peptide medium range SET 2

PFS2-A

C92H108O30

1692.693

14063

12860

PFS2-B

C121H140O40

2232.892

17236

18675

PFS2-C

C150H172O50

2773.092

19424

24137

PFS2-D

C184H214O61

3399.364

16623

23804

Peptide high range SET 3

PFS3-A

C194H228O66

3613.448

10885

17591

PFS3-B

C257H300O88

4793.900

13387

20419

PFS3-C

C320H372O110

5974.352

19046

23848

PFS3-D

C388H454O133

7240.876

19855

24677

Protein low range SET 4

PFS4-A

C398H468O136

7454.960

1290

13537

PFS4-B

C529H620O184

9915.916

1477

10635

PFS4-C

C660H772O230

12,376.87

1686

26134

PFS4-D

C796H934O277

14,923.90

1746

23936

Protein medium range SET 5

PFS5-A

C806H948O282

15,137.98

2071

2145

PFS5-B

C1073H1260O376

20,159.95

2292

2375

PFS5-C

C1340H1572O470

25,181.41

2493

2429

PFS5-D

C1612H1894O565

30,289.95

2182

2844

In order to provide numerous evenly spaced masses as calibration points, four different dendrimers were included in each of the five calibration sets, spanning multiple mass ranges: PFS1 (m/z 700–1600), PFS2 (m/z 1600–3500), PFS3 (m/z 3500–7500), PFS4 (m/z 7500–15,000), and PFS5 (m/z 15,000–30,000) (Figure 3).

3.3 Matrix and Solvent Compatibility

For internal calibration, the compatibility of the calibrant with a diversity of matrices and solvents is critical for broad utility. Because of the globular shape and highly branched structure of these dendrimers, they exhibit substantially enhanced solubility across a wide range of solvents compared with linear polymer analogs [21]. This reduced crystallinity is believed to be a major factor in the exceptional matrix compatibility observed for these dendrimers. Each one of the matrices investigated yielded a strong signal that allowed a simple, rapid calibration using the standard preparation described in the “Experimental” section (Supplementary Figure S2). Fourteen traditional matrices were included in the study: 6-aza-2-thiothymine (ATT), trans-2-[3-(4-tert-butylphenyl)-2-methyl-2-propenylidene] malonitrile (DCTB), α-cyano-4-hydroxycinnamic acid (CHCA), 1,5-diaminonaphthalene (DAN), dihydroxyacetophenone (DHAP), 2,5-dihydroxybenzoic acid (DHB), dithranol (DIT), ferulic acid (FA), galvinoxyl free radical (GFA), 2-(4-hydroxyphenylazo)benzoic acid (HABA), trans-indoleacrylic acid (IAA), mercaptobenzothiazole (MTB), 9-nitroanthracene (9-NA), and sinapic acid (SA). In addition, this calibration platform was equally compatible with the use of graphite as a matrix [22] and the recently commercialized “matrix-free” nanostructure assisted laser desorption/ionization (NALDI) [23] technique using silicon nanowires as a matrix substitute [24].

Likewise, the dendritic calibrants exhibited exceptional compatibility with a range of solvents. In the case of 9-nitroanthracene, calibration-quality spectra could be readily obtained by spotting samples from solutions of hexane, dichloromethane, chloroform, ethyl acetate, acetone, methanol, ethanol, acetonitrile, and dimethyl sulfoxide (Supplementary Figure S3). Using the 9-nitroanthracene matrix, spectra could also be obtained with 50 % water in acetonitrile, and 67 % water in acetonitrile, though the compatibility with larger proportions of water were limited by the insolubility of this matrix in 100 % water. In contrast, more conventional calibrants such as insulin or PEG exhibit compatibility with only a limited number of matrices (see “Supporting Information”) and have reduced solvent compatibility, especially with nonpolar solvents.

3.4 Calibrant Stability

Perhaps the most problematic shortcoming of the traditional peptide and protein calibrants is their limited stability both in solution and in bulk, requiring their storage at reduced temperature, typically at –4 to –20 °C. In contrast, SpheriCal calibrants exhibit exceptional shelf-lives at room temperature, even when exposed to ambient light and oxygen. To evaluate their stability, SpheriCal calibrants were stored (1) as a pure dry powder, (2) as a dry powder premixed with matrix (9-nitroanthracene) and cation source (sodium trifluoroacetate), and (3) as a solution (in acetonitrile) with matrix and cation source. When stored as a dry powder, at room temperature, the Protein Medium Range calibrant (PFS2) exhibited no visible sign of degradation even after 1605 d (~4.4 y) (Supplementary Figure S4a). This unprecedented stability enabled the calibrant to be premixed with the appropriate ratios of the matrix (9-nitroanthracene) and cation source (sodium trifluoroacetate) in dry form. Similarly, the premixed formulation also exhibited exceptional stability and reproducible spectra over a 3-year period (Supplementary Figure S4b) confirming that the calibrant can be stored long-term as a premixed formulation, substantially reducing the time and effort needed to prepare calibrant if frequent calibration is required. Finally, the stability of the solution-phase premixed formulation was also explored with acetonitrile as solvent, and the PFS2 calibrant exhibited stability in excess of 1 mo under these conditions (Supplementary Figure S3c), and even longer times for less polar solvents. Examination of peptide calibrants under these same conditions show reduced resolution, or a loss of signal in this same time frame (see “Supporting Information”).

3.5 Internal Calibration of Proteins

Because the ester linkages of the dendrimers are susceptible to complex formation with adventitious cations, including sodium and potassium, the above described external calibration studies have been optimized with an excess of sodium cation to minimize the signal from potassium or other cations. However, for internal calibration of peptides, the addition of sodium is not necessary. If sodium trifluoroacetate is used for the internal calibration of ATCH fragment (18–39), a series of sodiated carboxylate residues are observed because of the five carboxylate residues present, while each calibrant will exhibit exclusively the [M + Na]+ signal (Figure 4a). On the other hand, internal calibration without any added cation yields two signals per calibrant, [M + Na] + and [M + K] +, while only two major signals are observed for ATCH fragment (18–39): [M + H] + and [M + Na] + (Figure 4b).
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Figure 4

Internal calibration of ACTH fragment (18–39) with PFS2 using α-cyano-4-hydroxycinnamic acid as matrix with either (a) additional sodium trifluoroacetate (above) or (b) without any addition of cation sources (below)

4 Conclusion

The reliance upon peptides and proteins for mass spectral calibration in the high molecular weight range was largely a consequence of the lack of monodisperse alternatives during the early evolution of high mass MS techniques. However, many of these biological macromolecules suffer from poorly resolved signals, short shelf-lives, restricted matrix compatibility, and limited selection of masses, especially in the higher molecular weight ranges, making it difficult to obtain an accurate multi-point high mass calibration curve. With the optimization of SpheriCal, a polyester dendrimer-based calibration system, multiple advantages were observed, including multi-year shelf-lives in ambient conditions, unprecedented matrix compatibility, amenability to long-term storage after formulation, exceptional well-resolved signals, and an evenly spaced series of calibration points with masses as high as m/z 30,000. Considering the recent improvements in MS instrumentation for these higher molecular weight ranges, the parallel development of alternative high mass calibration systems is timely and critical for continued advances in high molecular weight mass spectrometry. The appropriateness of these compounds for MS/MS calibration of peptides was not addressed in this study; however, tandem MS experiments are currently under investigation.

Acknowledgments

This work was performed with the help of Tulane University and the Swedish Governmental Agency for Innovation Systems, VINNOVA, through the Forska&Väx program.

Supplementary material

13361_2013_777_MOESM1_ESM.pdf (1.5 mb)
ESM 1(PDF 1490 kb)

Copyright information

© American Society for Mass Spectrometry 2013