Angewandte
Chemie
DOI: 10.1002/anie.201407669
Protein Extraction from Tissue
Ultrafast Extraction of Proteins from Tissues Using Desorption by
Impulsive Vibrational Excitation**
Marcel Kwiatkowski, Marcus Wurlitzer, Maryam Omidi, Ling Ren, Sebastian Kruber,
Refat Nimer, Wesley D. Robertson, Andrea Horst, R. J. Dwayne Miller, and Hartmut Schlꢀter*
Abstract: A picosecond IR laser (PIRL) can be used to blast
proteins out of tissues through desorption by impulsive
excitation (DIVE) of intramolecular vibrational states of
water molecules in the cell in less than a millisecond. With
PIRL-DIVE proteins covering a range of a few kDa up to
several MDa are extracted in high quantities compared to
conventional approaches. The chemical composition of
extracted proteins remains unaltered and even enzymatic
activities are maintained.
molecules. This can be achieved by specific centrifugation and
precipitation methods or a combination of both.[4]
Until now, there have been few methods for extracting
a broad range of proteins with different chemical properties
from intact tissue samples in a single experimental step. Most
protocols are specific for the extraction of a distinct group of
proteins from a defined tissue type.[5–7] Thus, it would be
highly desirable to develop a sample preparation procedure
that is capable of extracting proteins with a broad range of
chemical properties in high yields, and which is soft and fast
such that activities are maintained and the conversion of
proteins by enzymatic or chemical reactions is minimized.
Romano and Levis showed that it is possible to ablate
high-mass, single-stranded DNA intact with a UV laser using
a chromophore and capture the ablated DNA for further
analysis.[8] Some other groups use laser ablation sample
transfer (LAST) prior to MALDI MS imaging (MALDI-
MSI) or ESI-MS analysis.[9–14] However, in the above-men-
tioned studies proteins were not directly extracted from intact
tissues by laser ablation. With MALDI-MSI it is possible to
desorb and analyze proteins and peptides from intact tissue
samples.[15,16] A detailed discussion is given in the Supporting
Information.
Recently, a new concept in laser surgery has been
developed by the Miller group based on a picosecond infrared
laser (PIRL) specifically tuned to the strong OH vibration
stretching band in water to drive ablation processes faster
than nucleation growth or energy transfer to adjacent tissue.
This method has been found to ablate entire proteins intact
into the gas phase.[17] The key enabling feature is the
elimination of unrestricted nucleation growth in the laser-
driven phase transition, which otherwise leads to cavitation-
induced shock waves and massive damage to surrounding
tissues and disintegration of proteins.[18–20] The ablation
process is made to happen faster than even acoustic transfer
of energy to adjacent tissue and strong acoustic attenuation of
the excited acoustics in the 100 GHz range also contributes to
the ablation process.[18] All the absorbed energy is converted
into translational degrees of freedom rather than being lost to
surrounding tissue through thermal or acoustic transport.
Most importantly, the ablation process occurs on timescales
faster than even collisional exchange of the excited water
molecules with the constituent proteins, which avoids thermal
fragmentation in the ablation step. Here, it needs to be fully
appreciated that liquid water couples vibrational energy
directly to translational motions, the very motions leading to
ablation. This transduction of the absorbed energy into
translational motions occurs on the 100 fs timescale, nearly
100 times faster than any other material with respect to
T
he extraction of proteins from biological tissues is a critical,
difficult, and time-consuming step in protein analysis. Pro-
teins have to be released from their native environments,
which requires a breakup of the outer cell membrane,
intracellular membranes, and surrounding extracellular struc-
tures. This is usually achieved by the application of a homog-
enization technique, which varies depending on the nature of
the tissue sample. In most cases, the first step includes cutting
the tissue into small pieces with sharp blades or a meat
grinder. Small pieces of soft tissue samples (brain, liver) can
be disrupted by ultrasonic homogenizers.[1] Hard and fila-
mentous tissues such as muscles, bones, and cartilage are
disrupted by mechanical homogenizers.[2] The choice of an
appropriate buffer system is important for an efficient protein
extraction. The buffer usually guarantees a stable pH since
deviation from the physiological pH may have denaturing
effects on proteins. Buffer systems may require additives to
stabilize proteins, protein–protein interactions, and enzymes.
However, extraction buffers should not result in chemical
reactions of the constituent proteins of the tissue.[3] After cell
disruption, large particles like cell debris, large organelles,
and insoluble compounds must be removed and proteins must
be separated from other biomolecules and small organic
[*] M. Kwiatkowski, M. Wurlitzer, M. Omidi, R. Nimer,
Priv.-Doz. Dr. A. Horst, Prof. Dr. H. Schlꢀter
University Medical Center Hamburg-Eppendorf
Department of Clinical Chemistry
Martinistrasse 52, Hamburg, 20246 (Germany)
E-mail: h.schlueter@uke.de
L. Ren, S. Kruber, Dr. W. D. Robertson, Prof. Dr. R. J. D. Miller
Max Planck Institute for the Structure and Dynamics of Matter
CFEL (Building 99)
Luruper Chaussee 149, 22761 Hamburg (Germany)
[**] We thank the SUREPIRL project (ERC advanced grant) and the
Virtual Institute “In vivo studies of biodegradable magnesium
based implant materials” MetBioMat (Helmholtz Society) for
financial support.
Supporting information for this article is available on the WWW
Angew. Chem. Int. Ed. 2015, 54, 285 –288
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
285