Direct analysis of reversed-phase high-performance thin layer chromatography separated tryptic protein digests using a liquid microjunction surface sampling probe/electrospray ionization mass spectrometry system

Article abstract of DOI:10.1255/ejms.1041

The sampling, ionization and detection of tryptic peptides separated in one-dimension on reversed-phase high-performance thin layer chromatography (HPTLC) plates was performed using liquid microjunction surface sampling probe electrospray ionization mass spectrometry. Tryptic digests of five proteins [cytochrome c, myoglobin, beta-casein, lysozyme and bovine serum albumin (BSA)] were spotted on reversed phase HPTLC RP-8 F254s and HPTLC RP-18 F254s plates. The plates were then developed using 70/30 methanol/water with 0.1 M ammonium acetate. A dual purpose extraction/electrospray solution containing 70/30/0.1 water/methanol/formic acid was infused through the sampling probe during analysis of the developed lanes. Both full scan mass spectra and data dependent tandem mass spectra were acquired for each development lane to detect and verify the peptide distributions. Data dependent tandem mass spectra provided both protein identification and sequence coverage information. Highest sequence coverages were achieved for cytochrome c and myoglobin (62.5% and 58.3%, respectively) on reversed phase RP-8 plates. While the tryptic peptides were separated enough for identification, the peptide bands did show some overlap with most peptides located in the lower half of the development lane. Proteins whose peptides were more separated gave higher sequence coverage. Larger proteins such as beta-casein and BSA which were spotted in lower relative amounts gave much lower sequence coverage than the smaller proteins. US Government 2009 All rights reserved.

Full text of DOI:10.1255/ejms.1041

J.F. Emory et al., Eur. J. Mass Spectrom. 16, 21–33 (2010)
21
received: 23 May 2009
■
revised: 24 June 2009 ■ Accepted: 25 June 2009 ■ Publication: 10 November 2009
EuroPEAN
JourNAL
of
MASS
SPEctroMEtry
Honouring Andries P. Bruins on the occasion of his 65th birthday
Direct analysis of reversed-phase high-
performance thin layer chromatography
separated tryptic protein digests using
a liquid microjunction surface sampling
probe/electrospray ionization mass
spectrometry system
Joshua F. Emory,a Matthew J. Walworth,a,b Gary J. Van Berkel,a,* Michael Schulzc and Susanne Minarikc
aorganic and Biological Mass Spectrometry Group, chemical Sciences Division, oak ridge National Laboratory, oak ridge, tN 37831-6131,
uSA. E-mail: vanberkelgj@ornl.gov
bDepartment of chemistry, university of tennessee, Knoxville, tennessee 37996-1600, uSA
cthin-Layer chromatography Laboratory, Performance and Life Science chemicals, Merck KGaA, 64293 Darmstadt, Germany
The sampling, ionization and detection of tryptic peptides separated in one-dimension on reversed-phase high-performance thin
layer chromatography (HPTLC) plates was performed using liquid microjunction surface sampling probe electrospray ionization
mass spectrometry. Tryptic digests of five proteins [cytochrome c, myoglobin, beta-casein, lysozyme and bovine serum albumin
(BSA)] were spotted on reversed phase HPTLC RP-8 F254s and HPTLC RP-18 F254s plates. The plates were then developed using
70/30 methanol/water with 0.1M ammonium acetate. A dual purpose extraction/electrospray solution containing 70/30/0.1
water/methanol/formic acid was infused through the sampling probe during analysis of the developed lanes. Both full scan mass
spectra and data dependent tandem mass spectra were acquired for each development lane to detect and verify the peptide dis-
tributions. Data dependent tandem mass spectra provided both protein identification and sequence coverage information. Highest
sequence coverages were achieved for cytochrome c and myoglobin (62.5% and 58.3%, respectively) on reversed phase RP-8 plates.
While the tryptic peptides were separated enough for identification, the peptide bands did show some overlap with most peptides
located in the lower half of the development lane. Proteins whose peptides were more separated gave higher sequence coverage.
Larger proteins such as beta-casein and BSA which were spotted in lower relative amounts gave much lower sequence coverage
than the smaller proteins.
Keywords: surface sampling probe, reversed-phase, thin layer chromatography, HPTLC, peptides, tryptic digest, electrospray, mass
spectrometry
this manuscript has been authored by a contractor of the uS Government under contract DE-Ac05-00or22725. Accordingly, the uS
Government retains a paid-up, non-exclusive, irrevocable, worldwide license to publish or reproduce the published form of this contribution,
prepare derivative works, distribute copies to the public, and perform publicly and display publicly, or allow others to do so, for uS Government
purposes.
ISSN: 1469-0667
doi: 10.1255/ejms.1041
© uS Government 2009
All rights reserved

22
Analysis of Tryptic Protein Digests Using an LMJ-SSP/ESI-MS System
Introduction
Protein identification is commonly done via “bottom up”1,2
In this work, the LMJ-SSP is used to directly analyze peptides
approaches that use high-performance liquid chromatography- from tryptic protein digests separated on hydrophobic rP-8
tandem mass spectrometry (HPLc-MS/MS) to identify and
characterize enzymatically-digested proteins. Peptide ions are
and rP-18 HPtLc plates. the results are compared and
contrasted with our previous work which used DESI to analyze
commonly generated from protein digests by either matrix- the same tryptic protein digests separated on NP HPtLc
assisted laser desorption/ionization (MALDI)3,4 or electro- plates. the separations on the rP HPtLc plates had lower
spray ionization (ESI).5 However, use of ESI is often preferred
because ESI produces multiply charged peptides that typically
provide more informative MS/MS data than singly charged
peptides.6,7 While HPLc methods are unlikely to be supplanted
by high-performance thin layer chromatography (HPtLc) for
peptide separations, HPLc of complicated peptide mixtures
can be time intensive and can consume significant amounts of
solvent. thin-layer separation methods present the ability to
separate several samples in parallel, archive the separations
and offer the possibility to analyze an individual separation
band resolution when compared to separations performed on
NP plates. Nonetheless, the overall protein sequence coverage
obtained from the rP plates via LMJ-SSP/ES-MS/MS analysis
were similar to that obtained from DESI-MS/MS analysis of
NP HPtLc plates. Proteins cytochome c. and myoglobin gave
the highest sequence coverage. the rP-8 plates separated
the tryptic peptides slightly better, contributing to the higher
sequence coverage obtained with these plates compared to
the rP-18 plates. overall, this report demonstrates that the
LMJ-SSP/ESI-MS system provides a viable means for mass
several times. Low detection levels and molecular identifica- spectrometric readout of peptides separated on hydrophobic
tion capabilities8–11 make mass spectrometry the analytical
method of choice for the readout of peptides separated on
thin layer chromatography (tLc) plates. MALDI-MS has been
used for the analysis of peptides and proteins from monolithic
tLc plates.12 recently, the ambient surface sampling ioniza-
tion technique desorption electrospray ionization-MS (DESI-
MS)13–17 has been used in our laboratory with a high degree of
success to analyze tryptic digests of five model proteins that
were separated in one18 or two dimensions19 on Proteochrom
HPtLc Silica gel 60 f254s and Proteochrom HPtLc cellulose
plates (hereafter referred to as normal phase (NP) plates).
Although DESI-MS/MS was quite successful in the analysis
of peptides separated on NP HPtLc plates, we have not been
rP HPtLc plates.
Experimental
Materials
Ammonium bicarbonate, trypsin, Lichrosolv methanol and
HPLc-grade water were used for tLc and were obtained from
Merck KGaA (Darmstadt, Germany). Lc-MS grade chromosolv
solvents acetonitrile and water both with 0.1% formic acid
(v/v) were obtained from Sigma Aldrich (St Louis, MS, uSA)
for use with the LMJ-SSP. Proteins bovine cytochrome
c, equine myoglobin, beta-casein from bovine milk, bovine
as successful in the analysis of these same peptides sepa- serum albumin (BSA) and lysozyme from chicken egg white
rated on hydrophobic reversed phase (rP) HPtLc plates. In
our experience, these hydrophobic plates are destroyed during
the DESI process when using water as a spray solvent. While
it may be possible to find a solvent system that allows DESI
analysis of these hydrophobic HPtLc plates, we chose to
pursue an alternative readout method, a liquid microjunc-
tion surface sampling probe (LMJ-SSP)/ESI-MS system. the
LMJ-SSP, based on a design first described by Wachs and
Henion20 consists of a pair of coaxial tubes with space between
the inner and outer capillaries. Liquid flows down this annular
space to the surface to be sampled forming a wall-less liquid
microjunction with the surface, extracts the analyte from the
surface and then is drawn up in the inner tube from which
it is sprayed into the mass spectrometer via an electrospray
emitter source. Earlier demonstrations of the analytical utility
of this LMJ-SSP include the sampling and analysis of dried
drugs or proteins or solutions thereof from wells on microtiter
were obtained from Sigma Aldrich (fluka, Buchs, Switzerland).
Hydrophobic HPtLc rP-18 and HPtLc rP-8 plates for sepa-
ration of the tryptically digested proteins were acquired from
Merck KGaA (Darmstadt, Germany).
Tryptic digestion
Digestions of the five model proteins with trypsin were
performed by dissolving each protein in 25 mM ammonium
bicarbonate to a final protein concentration of 2µgµL–1. trypsin
was added to the protein buffer mixture so the trypsin:protein
molar ratio was 1:100 and the mixture was incubated for 15h
at 37°c.
Thin-layer chromatography
Application of the samples to the HPtLc plates was done
using an AtS 4 fully automated sample applicator from cAMAG
(Muttenz, Switzerland). A sample volume of 7µL was applied
in a 6mm band at a dosage speed of 50nLs–1 to give a total of
plates,20 drugs captured in solid-phase extraction cards,21
a
variety of dyes, inks, or pharmaceuticals on paper or sepa- 14µg of protein per band. Protein concentrations per band were
rated on hydrophobic reversed-phase (rP-8 and rP-18) thin- as follows: cytochrome c (1132pmol), myoglobin (824pmol),
layer chromatography plates,21–25 exogenous compounds from
lysozyme (979pmol), beta casein (560pmol) and BSA (21pmol).
thin tissue sections,26,27 and surface-deposited and affinity- All samples were applied 10mm from the bottom of the HPtLc
captured proteins.28
plate with the bands spaced 15 mm apart. Development of

J.F. Emory et al., Eur. J. Mass Spectrom. 16, 21–33 (2010)
23
HPtLc plates was performed in a flat-bottomed chamber 1. the 4000 QtrAP was controlled by Analyst software version
using 70/30v/v methanol/water solution with 0.1M ammo- 1.4.2. typical operating conditions consisted of an ESI voltage
nium acetate to develop the plates. the solvent front distance
was 50mm, which was achieved in 45–60 minutes. the plates
were stained with fluorescamine using a normal tLc sprayer
(Merck KGaA).
of 4.5kV, a curtain gas at 20 instrumental units and a declus-
tering potential of 100V. In both instruments the nebulizing
gas used for probe aspiration was adjusted to the necessary
level to balance the pumped flow (10µLmin–1) into the probe.
An elution solvent composition of 70 / 30 (v / v)
water/acetonitrile with 0.1% formic acid by volume was used
to extract the analyte peptides from the tLc plates and was
pumped into the probe emitter at a rate of 10µLmin–1 using a
1mL or 2.5mL syringe attached to a Harvard Syringe pump. An
approximately 27cm long section of PEEK tubing (127µm inner
diameter and 1/16 inch outer diameter) with an upstream
ground point was used to supply the elution solvent to the
probe/emitter.
An MS2000 robotic x, y, z platform (Applied Scientific
Instrumentation, Inc., Eugene, or, uSA) was used to hold and
maneuver the tLc plates relative to the stationary LMJ-SSP
for analysis. the original microscope slide holder supplied
with the stage was replaced with a home-built tLc plate
holder made from rigid, non-conductive polymer. this tLc
plate holder held the tLc plate in a 100×100×1mm milled out
groove via finger-tightened plastic screws with semi-circular
heads. the tLc plate was held in the vertical position and
perpendicular to the LMJ-SSP. the MS2000 platform could be
controlled manually by use of a joystick in the x and y direc-
tions and by use of a jog wheel for z-direction control for initial
alignment and LMJ formation. Sampling operations on both
Mass spectrometry
two mass spectrometers were used in this work, an
LcQ-DEcA (thermo Electron, San Jose, cA, uSA) and a
4000 QtrAP (MDS ScIEX, concord, ontario, canada). the
LcQ-DEcA was the same instrument used to analyze tryptic
digested proteins separated on NP tLc plates via DESI and
data dependent MS/MS, which provided the NP tLc protein
sequence coverages listed in this work. thus, the results from
DESI analysis of separated protein digests on NP tLc plates
and the results from LMJ-SSP analysis of protein digests
separated on hydrophobic rP plates could be more directly
compared. the 4000 QtrAP provided better full scan mass
spectra (EMS mode) than the LcQ, so the data from the 4000
QtrAP were used to construct the ion current profiles for the
peptides identified from each protein digest.
A detailed description of each instrument setup with the
LMJ-SSP has been described in detail elsewhere.21,22 While
the surface sampling probes used with the 4000 QtrAP and
the LcQ-DEcA have the same internal components, they
have different outer casings. the LMJ-SSP on the 4000
QtrAP was built from a MicroIonSpray II emitter (MDS
ScIEX) and was attached to the 4000 QtrAP via a modi- the LcQ and the 4000 QtrAP were monitored in the hori-
fied nanospray source. the LMJ-SSP on the LcQ-DEcA was
designed in a similar fashion to that described by Wachs and
Henion,20 and by us,21 and was attached to the instrument
via a modified LcQ-DEcA nanospray ion source (thermo
zontal and vertical planes by two Panasonic GP-Kr222 closed-
circuit cameras (Panansonic Matsushita Electric corporation
of America, Secaucus, NJ, uSA). the camera used to observe
the liquid microjunction during operation was equipped with
Electron). Both LMJ-SSP probes had the following dimen- an optem 70 XL zoom lenses (thales optem Inc., fairport, Ny,
sions: inner sprayer/emitter capillaries with a 254µm outer
diameter and a 127 µm inner diameter, and the sampling
end of the probe had an outer capillary with a 635µm outer
diameter and a 330µm inner diameter. the only difference in
the two LMJ-SSP probes was the length of their respective
inner capillaries (LcQ-DEcA=10cm long, 4000 QtrAP=8cm
long).
operation of the LcQ Deca ion trap was performed using
Xcalibur software version 1.3 and the typical instrument
parameters consisted of an ESI voltage of 4 kV, a capillary
voltage of 7 V, a tube lens voltage of –35 V and a capillary
temperature of 200°c. Automatic gain control was used for
all measurements and tandem mass spectra were acquired
by operating the instrument in data dependent mode so that
the three most abundant peaks within each full scan mass
spectrum would be subjected to tandem mass spectrometry
and with dynamic exclusion set to three so MS/MS would
uSA).
All the tLc plate lane scans were enabled by using soft-
ware29 written in-house to control the ASI 2000 stage. Before
scanning a lane, a liquid microjunction was created at a posi-
tion along the development lane below the spotting point by
manual adjustment of the jog wheel and joystick via the ASI
2000 control system. After making the liquid microjunction,
the mass spectrometer data acquisition process was initiated
simultaneous with tLc plate positioning software. the HPtLc
plate was typically moved 60mm at a scan speed of 45µms–1
or 27µms–1 and typical scan times for an individual tLc lane
were 22min or 35min, respectively. Both the instrument data
acquisition time and tLc lane scan time were set so that the
instrument would acquire data from the distance below the
spotting point to beyond the solvent front. When the scan and
data collection processes were finished, the liquid microjunc-
tion was broken by moving the stage away from the probe in
be performed on a certain peak a maximum of three times. the z-direction and then repositioning the stage for the next
the normalized collision energy was set to 35% and three
microscans were acquired for each spectrum over a product
ion range of m/z 200–2000. An example of the setup of the
LMJ-SSP with the LcQ-DEcA system can be seen in figure
lane scan using the jog wheel and joystick.
Identification of the peptides observed during the tLc lane
scans was performed by extracting the MS/MS spectra from
raw data files and converting them to MS2 file format.30 the

24
Analysis of Tryptic Protein Digests Using an LMJ-SSP/ESI-MS System
Figure 1. Schematic of liquid microjunction surface sampling probe ESI-MS/MS setup (not to scale) on the LCQ-DECA for analysis of
HPTLC plates. The left side of the probe (magnified section) was responsible for making the liquid microjunction on the TLC plate and
the solvent flow and electrospray process can be understood by viewing the diagram below the probe.
MS2 files were searched using the DBDigger31 proteomics data- picture one also notes that the majority of the tryptic peptides
base search program which used the MASPIc32 scoring scheme
and the DtASelect33 algorithm for filtering the MS2 files. the
from the five protein digests (cytochrome c, myoglobin, beta-
casein, lysozyme and BSA), on both plates, appear to be
DtASelect algorithm used a DcN of at least 0.08 and cross corre- located in the middle rf region of the tLc plates. the pres-
lation (Xcorr) scores of 20 (+1), 25 (+2) and 40 (+3).
ence of most peptides in the middle rf region suggests that
these peptide separations would benefit from HPtLc plates
with higher band capacities. Additionally, visual comparison
of the peptide separations on either of these hydrophobic rP
plates to those separations of the same digests on NP plates
(plates not shown)18 showed the NP plate separations to be
superior.
the sequence coverage determined from the LMJ-SSP/
ESI-MS/MS analysis of each development lane on both types
of plate is summarized in table 1. Included in the table for
Results and discussion
General observations
An example of both a stained rP-8 and rP-18 HPtLc plate
showing the separations of all five protein digests is presented
in figure 2. from a simple visual inspection, one could
observe that the bands on the rP-8 plates were narrower
and more separated than those on the rP-18 plate, appar- comparison are the sequence coverages obtained from
ently indicating better separation of the peptides. from this a silica gel plate and from a cellulose plate by DESI-MS/

J.F. Emory et al., Eur. J. Mass Spectrom. 16, 21–33 (2010)
25
C18
C8
LYS
MYO
CYT C. β-CAS
BSA
LYS
MYO
CYT C. β-CAS
BSA
Figure 2. Tryptic digests of lysozyme (LYS, 979pmol spotted), myoglobin (MYO, 824pmol spotted), cytochrome c (CYT C, 1132pmol spot-
ted) B-casein (B-CAS, 560pmol spotted), and bovine serum albumin (BSA, 21pmol spotted) separated on RP-8 (left) and RP-18 (right)
HPTLC plates and stained using fluorescamine.
MS.18 these data show that the sequence coverage from the
rP-8 plate was generally slightly better than the coverage
from the rP-18 plate. In both cases, however, the sequence
coverage was comparable to the results from NP plates using
DESI-MS/MS. one difference between the analysis of the rP
and NP plates was that the NP plates were scanned at a
100µms–1, whereas the rP plates were scanned at either a
45µms–1 or 27µms–1. the rP plates were scanned at a slower
speed to help compensate for the lower quality separations of
the rP plate by giving the mass spectrometer more time to
analyze the overlapping peptide bands. Scanning at a 27µms–1
gave the highest sequence coverage, but the sequence
coverage was typically only 3–5% higher than that obtained
from the 45µms–1 surface scan rate.
coverage on the rP plates than the other three proteins
lysozyme, beta casein and BSA. this resulted, in part, because
the digests from the proteins were applied in equal mass
amounts, not molar amounts. the sequence coverage was
lowest for BSA (21pmol) and beta-casein (560pmol), which
were applied in the lowest molar amounts. cytochrome c.
(1132 pmol), lysozyme (979pmol) and myoglobin (824pmol),
which were applied in much higher molar amounts, showed
the highest sequence coverage. It should also be noted that
the lane scans of the protein digests performed for this work
were approximately 0.5 mm wide and the band widths in
each tLc lane were 6mm wide. therefore, the LMJ-SSP was
only sampling from about 8% of the total band area on the
plate. this suggests that the LMJ-SSP analysis approach
(like DESI) might provide superior results with more concen-
trated compact peptide bands. furthermore, multiple surface
the summarized results in table 1 show that the proteins
cytochrome c and myoglobin give much higher sequence
Table 1. Sequence coverages obtained for five protein tryptic digests analyzed by LMJ-SSP/ESI-MS/MS for the RP-8 and RP-18 HPTLC plates
and by DESI-MS/MS for the silica gel (NP) and cellulose HPTLC plates.
LMJ-SSP
HPTLC RP-8 F254s
DESI18
ProteoChrom HPTLC
Protein
HPTLC P-18 F254s
8.6%
ProteoChrom HPTLC
Cellulose
Silica gel 60 F254s
BSA
5.3%
12.1%
62.5%
14.3%
8.6%
14.7%
69.2%
60.8%
50.4%
(66kDa, 21pmol)
Beta casein
(25kDa, 560pmol)
12.1%
17.4%
59.6%
66.0%
27.1%
cytochrome c
(12kDa, 1132pmol)
59.6%
Myoglobin
(17kDa, 824pmol)
54.2%
58.2%
45.7%
Lysozyme
34.1%
(14kDa, 979pmol)

26
Analysis of Tryptic Protein Digests Using an LMJ-SSP/ESI-MS System

J.F. Emory et al., Eur. J. Mass Spectrom. 16, 21–33 (2010)
27

28
Analysis of Tryptic Protein Digests Using an LMJ-SSP/ESI-MS System
scans of a single development lane could be accomplished to
improve confidence in peptide identification and to possibly
improve sequence coverage.
from cytochrome c, HPGDfGADAQGAM*tK from myoglobin,
EM*PfPK from beta-casein, and IVSDGNGM*NAW+VAW+r
and GtDVQAW+Ir from lysozyme. the abundance of the
oxidized peptides in some cases could be linked to time of
exposure of the plate to the laboratory environment.
Investigation of the full scan mass spectra acquired during
the surface scan of the development lanes of all five proteins
revealed expected tryptic peptide masses for these proteins
that were not identified by the MS/MS data-dependent scans
(for example, see tables 2 and 3). these full scan mass spectra
also showed the presence of sodium adducts at 15–30% of the
intensity of the protonated molecule for almost every major
peptide observed. Sodiated peptides were also observed
during the study of NP plates by DESI-MS. Sodium adducts
decrease the overall protonated molecule signal, which can
decrease the overall quality of the MS/MS data acquired from
the protonated molecule. time spent dissociating protonated
and sodiated versions of the same peptide might also cause
Cytochrome c
the LMJ-SSP/ESI-MS/MS analysis of cytochrome c digests
separated on rP-8 and rP-18 HPtLc plates resulted in
sequence coverages of 62.5% and 59.6%, respectively. these
results were consistent with the visual inspection of the
stained rP-8 and rP-18 tLc plates, which showed that the
rP-8 plate provided slightly better separations than the rP-18
plate. Approximate correlation of each peptide identified with
the bands in the development lane on the two plates is shown
by the extracted ion current profiles in figures 3 and 4. from
lower abundant peptides at a particular location in a devel- these figures one notes most of the peptides identified by MS/
opment lane to be overlooked in the data dependent MS/MS
experiments.
MS are located in a relatively narrow, ~10 mm distance, on
both the rP-8 and rP-18 plate. one peptide that was always
observed in high intensity on both the rP-8 and rP-18 plates,
but which never contributed to the sequence coverage, was
GItWGEEtLMEyLENPK which was located at 20.5 mm up the
devlopement lane. Both the unmodified and modified versions
of this peptide were identified from the LcQ full MS scan data
at m/z 1005 and m/z 1030, but a product ion spectrum adequate
for confident identification was not obtained. Doing so would
have increased the reported sequence coverage by ~10%. the
We also noted that several peptides from three of the
proteins investigated (beta casein, myoglobin, cytochrome
c and lysozyme) showed evidence of modifications including
oxidation and N-terminal acetylation. these modifications
needed to be included in the database searching parameters
to ensure identification of the peptides. In the text and tables
2 and 3, these modifications are indicated by a symbol placed
after the modified amino acid, where “*” and “+” indicate
single oxidation and double oxidation, respectively, and “:” modified version of this peptide contained a double oxidation
indicates N-terminal acetylation. Modified peptides observed
included M*IfAGIK, GItW+GEEtLM*EyLENPK, and G:DVEK
on tryptophan and a single oxidation on methionine, and the
relative intensity ratio of modified to unmodified appeared
2.5e8
GITWGEETLMEYLENPK
YIPGTK
IFVQK
TPGNLHGLFGR
TGQAPGFSYTDANK
G:DVEK
1.5e8
IFVQKCAQCHTVEK
EDLIAYLK
M*IFAGIK
5e7
0
10
20
30
40
50
mm
Figure 3. Extracted ion current profiles for all peptides identified by LMJ-SSP/ESI-MS/MS of a cytochrome c tryptic digest separated on
a RP-8 HPTLC plate. The GITWGEETLMEYLENPK peptide was only detected in the full scan MS.

J.F. Emory et al., Eur. J. Mass Spectrom. 16, 21–33 (2010)
29
2.0e8
TGQAPGFSYTDANK
YIPGTK
GITW+GEETLM*EYLENPK
IFVQK
KTQAPGFSYTDANK
MIFAGIK
G:DVEK
1.2e8
TPGNLHGLFGR
KATNE
EDLIAYLK
M*IFAGIK
4e7
0
10
20
30
40
50
mm
Figure 4. Extracted ion current profiles for all peptides identified by LMJ-SSP/ESI-MS/MS of a cytochrome c tryptic digest separated on
a RP-18 HPTLC plate. The GITW+GEETLM*EYLENPK peptide was only detected in the full scan MS.
to be proportional to the length of time the tLc plate was
exposed to air.
brightly stained band. Moreover, the amine group on the
lysine side chain has been reported to be inhibited in reac-
tion with fluorescamine.34
Details regarding the sequence coverage obtained for
cytochrome c analysis on rP-8 and rP-18 plates in this
work, as well as on NP plates and on cellulose plates using
DESI-MS/MS, are presented in table 2. this table shows that
the majority of the peptides were identified on both types of
rP plates as well as on the NP and cellulose plates. Another
similarity was that the peptide GItWGEEtLMEyLENPK was not
confirmed by MS/MS on any of the plates. Small peptides, under
500 Da, as well as peptides with low rf values and high numbers
of acidic or basic sites, were not confirmed by MS/MS.
Note that prior to mass spectrometric analysis it was
assumed that every stained band on a rP HPtLc plate
correlated with a peptide. However, this assumption may
Myoglobin
the LMJ-SSP/ESI-MS/MS analysis of tryptic digested
myoglobin on the rP-8 and rP-18 plates resulted in myoglobin
sequence coverages of 58% and 54%, respectively. As with
cytochrome c, higher sequence coverage was obtained from
analysis of the rP-8 plate, probably because the peptides
were separated slightly better and traveled further up the
rP-8 plate development lane. Extracted ion current profiles
of peptides observed from the myoglobin rP-8 and rP-18
HPtLc lanes are shown in figures 5 and 6. the slightly higher
sequence coverage associated with the rP-8 plate can be
now be questioned because there were several band loca- attributed to the identification of the ELGfQG peptide on the
tions on the tLc plate that did not produce ions that could
be attributed to an expected tryptic peptide (for cytochrome
c and the other proteins too). the species at these band
locations might be poorly extracted from the plate, might
rP-8 plate, but not on the rP-18 plate. this peptide, found at
43mm on the rP-8 plate, accounts for the majority of the ion
signal at that position, but is only identified by MS/MS once
on the rP-8 plate despite its high abundance. A close look
give poor response in ESI or might be unexpectedly modi- at the rP-18 myogobin data shows that the ELGfQG peptide
fied peptides. Also, peptides were identified that were not
visually observable on the plate. the relatively abundant
was targeted for MS/MS, but did not dissociate effectively,
making the MS/MS scores too low for confident identification
N-terminally-acetylated peptide G:DVEK from the cyto- by DBDigger. Another myoglobin peptide that was very visible
chrome c digest, for example, could not be definitively
correlated with a particular band even though it appeared
by itself in the development lane at high rf (see figures 3
and 4). N-terminal acetylation of this peptide prevents it
from reacting with the fluorescamine and appearing as a
in the full scan data, but not identified by MS/MS, was fDK at
m/z 409. this peptide was co-located at approximately 26mm
up the development lane (see figure 5) on the rP-8 myoglobin
plate with other peptides that were identified by MS/MS. this
same peptide was also observed in the full scan mass spectra

30
Analysis of Tryptic Protein Digests Using an LMJ-SSP/ESI-MS System
2.5e8
1.5e8
5e7
HGTVVLTALGGILK
LFTGHPETLEK
HPGDFGADAQGAMTK
ELGFQG
YLEFISDAIIHVLHSK
ALELFR
VEADIAGHGQEVLIR
FDK
NDIAAK
0
10
50
20
30
40
mm
Figure 5. Extracted ion current profiles for all peptides identified by LMJ-SSP/ESI-MS/MS of a myoglobin tryptic digest separated on a
RP-8 HPTLC plate.
by DESI-MS from silica gel and cellulose plates, but not identi- by only about 5mm on average (see figures 5 and 6). further
fied by MS/MS.
examination of the myoglobin data shows that a few of the
peptides identified from both the rP-8 and rP-18 tLc plates
were present at the spotting point as well as about halfway up
the developed lane (~27mm). this may have occurred because
these particular peptides are more hydrophobic than the other
Interestingly, the peptide NDIAAK traveled approximately
20 mm further up the rP-8 plate than it did on the rP-18
plate, whereas all the other peptides that were observed on
both the rP-8 and rP-18 plates differed in distance traveled
2.0e8
HGTVVLTALGGILK
YLEFISDAIIHVLHSK
1.2e8
YLEFISDAIIHVLHSK
LFTGHPETLEK
VEADIAGHGQEVLIR
VEADIAGHGQEVLIR
HPGDFGADAQGAMTK
ALELFR
NDIAAK
4e7
20
30
40
50
0
10
mm
Figure 6. Extracted ion current profiles for all peptides identified by LMJ-SSP/ESI-MS/MS of a myoglobin tryptic digest separated on
a RP-18 HPTLC plate.

J.F. Emory et al., Eur. J. Mass Spectrom. 16, 21–33 (2010)
31
myoglobin peptides. the presence of some peptides at the
tion peptide separations. the ability with the LMJ-SSP to
spotting point was also observed, albeit, to a lesser degree, analyze hydrophobic rP HPtLc plates makes it a comple-
for cytochrome c and BSA digests. this phenomenon was
not readily noticeable in the development lanes containing
lysozyme and beta casein. the reasons for this are unclear, but
may relate to the specific peptides present or phenomenon
related to the spotting process.
mentary ambient ionization method to DESI, which we have
found better suited to the analysis of NP HPtLc plates.
further advances in the quality of peptide separations on
NP and rP HPtLc plates will likely improve the protein
sequence coverage obtainable by these ambient surface
sampling/ionization techniques.
BSA, beta-casein and lysozyme
the LMJ-SSP/ESI-MS/MS analysis of tryptic digested
lysozyme, beta-casein and BSA on the rP-8 and rP-18 plates
provided results phenomenologically similar to those already
Acknowledgments
discussed for cytochrome c and myoglobin. once again, the J.f.E. acknowledges an oak ridge National Laboratory (orNL)
rP-8 separations for each of these three protein digests
were slightly better than rP-18 separations with the rP-8
mass spectral data providing a slightly higher sequence
coverage.
appointment through the orNL Postdoctoral research
Associates Program. Dr Julian Philips (thermo fisher
Scientific) is thanked for the loan of the LcQ DEcA mass spec-
trometer. Dr Vilmos Kertesz (orNL) is thanked for help in
setting up the bottom-up peptide/protein data analysis work
flow. the MicroIonSpray head used to fabricate one of the
surface sampling probes was provided through a cooperative
research and development agreement (crADA) with MDS Sciex
(orNL02-0662). this research was supported by the Battelle
Memorial Institute technology Maturation fund. the surface
scanning platform and associated control software used in
this study was developed with support from orNL technology
transfer and Economic Development (ttED) royalty funds.
orNL is managed and operated by ut-Battelle, LLc, for the
Although the separation of BSA peptides appeared
adequate on the rP plates, the relatively low molar amount
of BSA (21pmol) spotted on the plates compared to the other
protein digests may have contributed to the low sequence
coverage (< 10% for both rP-8 and rP-18 plates). As with
BSA, the smaller amount of beta-casein spotted (560 pmol)
on the plates probably contributed to the relatively low
sequence coverage for this protein as well (12.1% for both
rP-8 and rP-18 plates). the relatively narrow separation
range of the beta-casein peptides on the HPtLc plate, which
was 7.5mm for the rP-8 plate and 5mm for the rP-18 plate, united States Department of Energy under contract DE-Ac05-
also played a role in the low sequence coverage. factors that
influence the protein sequence coverage attainable from the
tryptic digests using this surface sampling analysis method
include efficiency of the tryptic digestion, average size of
the tryptic peptides produced from a protein, the ionization
efficiency of the peptides produced, the amount of material
on the plate and the separation efficiency of the peptides on
the tLc plate.
00or22725.
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