Optimized sample-processing time and peptide recovery for the mass spectrometric analysis of protein digests
Doris E. Terry
2
3
4
Edward Umstot
2
3
Dominic M. Desiderio
0
1
2
3
0
Also at the Department of Neurology, The University of Tennessee Center for Health Science
, Memphis,
TN 38163, USA
1
Also at the Department of Molecular Sciences, The University of Tennessee Center for Health Science
, Memphis,
TN 38163, USA
2
Published online April 2, 2004 Address reprint requests to Dr. D. E. Terry,
Department of Biochemistry, Purdue University, 175 S. University Street
,
West Lafayette, IN 47907, USA
3
The Charles B. Stout Neuroscience Mass Spectrometry Laboratory, The University of Tennessee Center for Health Science Memphis
,
Tennessee, USA
4
Current address: Bindley Bioscience Center
, Discovery Park,
Purdue University
,
West Lafayette, IN 47907, USA
Proteomics requires an optimized level of sample-processing, including a minimal sampleprocessing time and an optimal peptide recovery from protein digests, in order to maximize the percentage sequence coverage and to improve the accuracy of protein identification. The conventional methods of protein characterization from one-dimensional or two-dimensional gels include the destaining of an excised gel piece, followed by an overnight in-gel enzyme digestion. The aims of this study were to determine whether: (1) stained gels can be used without any destaining for trypsin digestion and mass spectrometry (MS); (2) tryptic peptides can be recovered from a matrix-assisted laser desorption/ionization (MALDI) target plate for a subsequent analysis with liquid chromatography (LC) coupled to an electrospray ionization (ESI) quadrupole ion trap MS; and (3) an overnight in-gel digestion is necessary for protein characterization with MS. These three strategies would significantly improve sample throughput. Cerebrospinal fluid (CSF) was the model biological fluid used to develop these methods. CSF was desalted by gel filtration, and CSF proteins were separated by two-dimensional gel electrophoresis (2DGE). Proteins were visualized with either silver, Coomassie, or Stains-All (counterstained with silver). None of the gels was destained. Protein spots were in-gel trypsin digested, the tryptic peptides were purified with ZipTip, and the peptides were analyzed with MALDI and ESI MS. Some of the samples that were spotted onto a wax-coated MALDI target plate were recovered and analyzed with ESI MS. All three types of stained gels were compatible with MALDI and ESI MS without any destaining. In-gel trypsin digestion can be performed in only 10 - 60 min for protein characterization with MS, the sample can be recovered from the MALDI target plate for use in ESI MS, and there was a 90% reduction in sample-processing time from overnight to ca. 3 h. (J Am Soc Mass Spectrom 2004, 15, 784 -794) 2004 American Society for Mass Spectrometry
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Ttion and identification from two-dimensional
he conventional method for protein
characteriza(2-D) gels involves the destaining of the excised
gel pieces, followed by an overnight in-gel enzyme
digestion and mass spectrometry (MS) [110]. Recently,
4 and 6 h in-gel trypsin digestions have been used for
mass spectrometry (MS) analysis [7, 11].
Protein identification with matrix-assisted laser
desorption/ionization-time of flight (MALDI-TOF) MS has
been enhanced by employing methods such as ZipTip
(Millipore Corp., Bedford, MA) purification [5, 1216] and
by the use of a hydrophobic sample support for sample
purification and sample delivery into the MS. The use of
sample supports has been developed to improve detection
sensitivity and to remove salts. Some of the polymer
materials that have been used for hydrophobic surfaces on
MALDI target plates include polytetrafluoroethylene
(Teflon; [4, 1719]), polyurethane [20], paraffin wax film [21],
and polyethylene and polypropylene [22]. For routine MS
analysis, a sample is processed for either MALDI or ESI
MS, or it is split for a MALDI and ESI MS analysis. There
is no published report on the recovery of tryptic peptides
from a MALDI target plate. The aims of this study were to
determine the compatibility of in-gel trypsin digestion of
stained protein spots with MS, to evaluate the time necessary
for in-gel trypsin digestion for protein characterization with
MS, and to recover tryptic peptides from a MALDI target
plate for subsequent analysis with ESI.
A human CSF sample was obtained after appropriate
institutional approvals by lumbar puncture (courtesy of
Dr N. M. Oyesiku, Emory University, Atlanta), frozen
immediately with liquid nitrogen, and shipped on dry
ice to Memphis. It was thawed, a cocktail of protease
inhibitors [10 mM EDTA, 1 mM phenylmethylsulfonyl
fluoride (PMSF), Complete mini (Roche, Mannheim,
Germany)] was added, and the CSF was stored ( 80 C)
until analyzed.
Desalting of Cerebrospinal Fluid
CSF samples were desalted by Bio-Spin
chromatography, as described by the manufacturer (Bio-Rad
Laboratories, Hercules, CA,). Bio-gel P-6 (30 g, Bio-Rad
Laboratories) was washed with the standard cycle of
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