are low throughput and typically requirethe sacrifice of the
cleaved material, thus reducing overall yields.
Previously, researchers have gone to great lengths to
install mixed linkers8 or to employ highly technical instru-
mentation (e.g., nanomanipulation and various MALDI
techniques)9 to extract information about substrates with-
out requiring global cleavage from the resin. In particular,
time-of-flight-secondary ion mass spectrometry (TOF-
SIMS) has been shown to be effective for ionization of
surface associated peptides from polystyrene beads. This
method can be coupled with the release of peptides from
solid supports with TFA vapors, but failed to yield analyte
ions when extended to covalently bound substrates.10
Photocleavable linkers that are activated by short pulses
from MALDI lasers have also been used to simultaneously
remove and analyze substrates from resin based solid
supports.11 In addition, there are several recent reports
that employ electron impact mass spectrometry to the
analysis of polystyrene resins bearing simple surface
functionalizations.12 We now present the first example of
the use of direct analysis in real time mass spectrometry
(DART-MS) for the analysis of resin bound substrates and
demonstrate that this technique is appropriate for a diverse
array of SPPS and SPOS products. The method can be
adapted to determine peptide sequence from MS fragmen-
tation studies and can also be used to monitor reaction
progress in SPOS systems.
The field of mass spectrometry is currently undergoing a
period of intense technological advancement, centered on
the design and development of new ionization methods.
Among these new techniques, desorption electrospray
ionization (DESI) and DART have emerged as two new
approaches for the analysis of heterogeneous substrates
under atmospheric pressure conditions. Together with
advances in TOF-SIMS13 and MALDI, these techniques
now offer the ability to analyze a diverse array of hetero-
geneous substrates, from mass spectral imaging of cryosec-
tioned biological samples,14 to the detection of explosives
residue on clothing.15
Figure 1. Schematic of DART ionization source.
formed by atmospheric pressure glow discharge, with
water molecules within the atmospheric pressure region
of the ionization source, to form protonated water dimers
(Figure 1). Subsequent proton transfer from these water
dimers to analyte molecules affords analyte ions that can
be detected using standard mass analyzer technologies.16
The principle advantages of DART ionization over other
techniques are the high degree of tolerance for sample
heterogeneity, and elimination of requirement for sample
preparation. This provides an opportunity for the investi-
gation of materials that have previously been inaccessible
to mass spectrometric analysis. Given the recent rash of
reports detailing heterogeneous substrate analysis using
DART, we were motivated to explore the potential for
using this technology to directly analyze resin-bound pep-
tides without prior chemical cleavage.
Our initial study was performed with a small (15 μM)
solid phase synthesis lantern, containing a peptide scaffold
of relevance to our global health drug discovery program.17
The washed lantern (3ꢀ DMF, 3ꢀ CH2Cl2, 3ꢀ DMF) was
exposed to the ionization region of the source, and gave
immediate strong signals for both [M ꢁ Fmoc þ H]þ and
various y ions (Supporting Information, Figure S12),
showing that it was possible to readily observe predicted
protonated signals for covalently attached analytes using
DART-MS. Encouraged by this initial success, we ex-
panded the scope of the study to include both the most
commonly used polymer supports (SPPS resins, lanterns,
and TentaGel), and the most frequently used linkers (trityl,
Rink amide, Wang, and Merrifield). The same substrate
(Fmoc-VVVAF-resin) was synthesized on each of the solid
supports using standard SPPS methods (Supporting
Information), and analyzed by DART-MS under identical
conditions. In total we synthesized 12 substrates for analysis,
containing different combinations of solid supports and
linkers. These included four polystyrene-based resin ana-
logues, three TentaGel samples, and two lantern analo-
gues. In all cases we were able to observe either [M þ H]þ
or [M ꢁ Fmoc þ H]þ ions, indicating that cleavage and
ionization of analytes is independent of both polymer
support and linker composition.
In DART analysis, ionization is achieved through the
collision of metastable excited-state neutral helium atoms,
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Verbeck, G. F.; Petros, R. A. Anal. Biochem. 2010, 398, 7–14. (b)
Greving, M. P.; Kumar, P.; Zhao, Z. G.; Woodbury, N. W. Langmuir
2010, 26, 1456–1459.
(10) Brummel, C. L.; Lee, I. N.; Zhou, Y.; Benkovic, S. J.; Winograd,
N. Science 1994, 264, 399–402.
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820. (b) Semmler, A.; Weber, R.; Przybylski, M.; Wittmann, V. J. Am.
Soc. Mass. Spectrom. 2009, 21, 215–219.
(12) (a) Heinze, K.; Winterhalter, U.; Jannack, T. Chem.;Eur. J.
2000, 6, 4203–4210. (b) Chavez, D.; Ochoa, A.; Madrigal, D.; Castillo,
M.; Espinoza, K.; Gonzalez, T.; Velez, E.; Melendez, J.; Garcia, J. D.;
Rivero, I. A. J. Comb. Chem. 2003, 5, 149–154.
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Kim, H. S. Appl. Surf. Sci. 2008, 255, 1064–1067. (b) Min, H.; Kim, Y.;
Yu, H.; Moon, D. W.; Lim, S. J.; Yoon, H. J.; Lee, T. G.; Shin, S. K.
Chem.;Eur. J. 2008, 14, 8461–8464.
(14) Esquenazi, E.; Yang, Y. L.; Watrous, J.; Gerwick, W. H.;
Dorrestein, P. C. Nat. Prod. Rep. 2009, 26, 1521–1534.
(15) Cody, R. B.; Laramee, J. A.; Durst, H. D. Anal. Chem. 2005, 77,
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