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The LC-MS Workflow: Is MALDI or ESI the Preferable Ionization Method?
Anders Tangen,*a Hanno Ehring,a John Flensburg,a Maria C. Prieto Conawayb and Henrik Wadenstena
aAmersham Biosciences AB, a GE Healthcare Company, Björkgatan 30, SE-751 84 Uppsala, Sweden
bThermo Electron Corp. 355 River Oaks Parkway, San Jose, CA, USA, 95134
µLC Configuration
Introduction
The use of the LC-MS workflow as a complement to 2DE-MS
workflow is growing in popularity.1, 2 Some of the 2DE limitations
are more easily overcome with the use of LC (hydrophobic or
extremely small/large or basic proteins), in part because the typical
sample is a tryptic digest – not a full length protein. Traditionally,
ESI mass spectrometers have been used as detectors for LC, due
to the simplicity of the interface. Today, many researchers also do
LC with MALDI-MS detection, either as a complement or an
alternative to LC-ESI-MS. Analysis speed and sensitivity are the
most frequently used arguments for the proper choice of instrument
configuration. However, from theoretical considerations one could
argue that the two ionization techniques are complementary by
nature.
Methods
MALDI-TOF, ESI-TOF, MALDI-LTQ and ESI-LTQ instruments have
been evaluated as detectors for the LC-MS workflow. Parameters
such as sensitivity, analysis time, ease of use and applicability for
peptides with different physiochemical characteristics have been
considered. Multivariate analysis has been used to find correlations
between fundamental peptide characteristics and probability of ESI-
MS or MALDI-MS detection.
Summary
Higher sequence coverage is obtained with the use of ESI.
Compared to ESI, relative MALDI detectability is correlated with
larger, more hydrophobic peptides, having lower pI values. The
optimum choice of MS detector is also depending on the type of
application.
µLC-ESI-TOF vs. MALDI-TOF:
Which peptides are detected?
MALDI only: 8 peptides
ESI & MALDI common: 15 peptides
LC-ESI only: 18 peptides
Total: 41 peptides detected (ESI + MALDI)
Correlation Between Peptide
Characteristics and ESI or MALDI
Detectability
The following peptide parameters were tested for influence upon
ESI and MALDI detectability: Hydrophobicity, pI, mass, amino acid
coverage and cystein content
PLS Loading Map (Only detected peptides included in model)
-”Maximum correlation between X- and Y-variables”
Comments to Relative Detectability
Compared to MALDI-TOF, µLC-ESI-TOF detectability is correlated
with smaller peptides having higher pI values and lower RPC
retention times.
Compared to µLC-ESI-TOF, MALDI-TOF detectability is correlated
with larger peptides, having lower pI values and higher RPC
retention times.
For a tryptic digest of BSA, most of the cysteines are located in the
larger peptides. In general, the larger peptides are harder to detect.
Experimental Conditions
Samples.
For ESI-TOF and MALDI-TOF experiments :
Tryptic digest of Bovine Serum Albumin (Michrom, USA).
For ESI-LTQ and MALDI-LTQ experiments:
Tryptic Digest (LC Packings, USA) of 6 Proteins (Cytochrome c,
Lysozyme, Alcohol dehydrogenase, Bovine serum albumin, Apo-
transferrin, β-Galactosidase), lyophilized, CAF modified using
standard protocol.
Chromatography Systems.
For ESI-TOF and MALDI-TOF experiments :
Ettan™ microLC (Amersham Biosciences, UK).
For ESI-LTQ and MALDI-LTQ experiments:
Ettan™ MDLC (Amersham Biosciences, UK). Controlled by
Xcalibur (Thermo Electron, USA) 1.4 via the VI interface.
MDLC dimension 1:
Column: 0.3 x 30mm BioBasic SCX, 5 µm, 300 Å (Thermo Electron,
USA). Mobile phase: 0.1% FA in H2O. Flow Rate: 10 µl/min
Flow through fraction analysed
MDLC dimension 2:
Trap column: 0.3 x 5mm, PepMap C18, 5µm, 100Å (Dionex/LC
Packings, USA). RPC column: 75µm x 150mm, PepMap C18, 3µm,
100Å (Dionex/LC Packings, USA). Buffer A: 0.1% formic acid.
Buffer B: 0.1% formic acid in 84% ACN. Gradient: 0–40% B in 20
min. Flow rate: 300 nl/min
MDLC-MALDI Interface: Probot (Dionex/LC Packings, USA).
MS.
For ESI-TOF and MALDI-TOF experiments :
Enterprise ESI-TOF (Analytica of Branford, USA).
Ettan™ MALDI-ToF (Amersham Biosciences, UK).
For ESI-LTQ and MALDI-LTQ experiments:
Finnigan ESI-LTQ and Finnigan vMALDI-LTQ (Thermo Electron,
USA). Dynamic exclusion activated
Multivariate Analysis.
SIMCA-P 8.0 (Umetrics, Sweden)
Phe-Gly-Glu-Arg-
Val-Ala-Ser-Leu-Arg-
Gln-Arg-Leu-Arg-
Ala-Phe-Asp-Glu-Lys-
Gly-Val-Phe-Arg-Arg-
Lys-Phe-Trp-Gly-Lys-
Ala-Trp-Ser-Val-Ala-Arg-
Ser-Glu-Ile-Ala-His-Arg-
Leu-Val-Thr-Asp-Leu-Thr-Lys-
Tyr-Leu-Tyr-Glu-Ile-Ala-Arg-
Asp-Leu-Gly-Glu-Glu-His-Phe-Lys-
Ser-His-Cys-Ile-Ala-Glu-Val-Glu-Lys-
Cys-Cys-Thr-Glu-Ser-Leu-Val-Asn-Arg-
Leu-Val-Asn-Glu-Leu-Thr-Glu-Phe-Ala-Lys-
Cys-Cys-Thr-Lys-Pro-Glu-Ser-Glu-Arg-
Phe-Lys-Asp-Leu-Gly-Glu-Glu-His-Phe-Lys-
His-Leu-Val-Asp-Glu-Pro-Gln-Asn-Leu-Ile-Lys-
Ser-Leu-His-Thr-Leu-Phe-Gly-Asp-Glu-Leu-Cys-Lys-
Arg-His-Pro-Glu-Tyr-Ala-Val-Ser-Val-Leu-Leu-Arg-
Tyr-Ile-Cys-Asp-Asn-Gln-Asp-Thr-Ile-Ser-Ser-Lys-
Thr-Cys-Val-Ala-Asp-Glu-Ser-His-Ala-Gly-Cys-Glu-Lys-
Leu-Arg-Cys-Ala-Ser-Ile-Gln-Lys-Phe-Gly-Glu-Arg-
Leu-Gly-Glu-Tyr-Gly-Phe-Gln-Asn-Ala-Leu-Ile-Val-Arg-
Leu-Lys-Glu-Cys-Cys-Asp-Lys-Pro-Leu-Leu-Glu-Lys-
Asp-Asp-Pro-His-Ala-Cys-Tyr-Ser-Thr-Val-Phe-Asp-Lys-
Asp-Ala-Phe-Leu-Gly-Ser-Phe-Leu-Tyr-Glu-Tyr-Ser-Arg-
Leu-Lys-Pro-Asp-Pro-Asn-Thr-Leu-Cys-Asp-Glu-Phe-Lys-
Lys-Val-Pro-Gln-Val-Ser-Thr-Pro-Thr-Leu-Val-Glu-Val-Ser-Arg-
Met-Pro-Cys-Thr-Glu-Asp-Tyr-Leu-Ser-Leu-Ile-Leu-Asn-Arg-
Tyr-Asn-Gly-Val-Phe-Gln-Glu-Cys-Cys-Gln-Ala-Glu-Asp-Lys-
Arg-Pro-Cys-Phe-Ser-Ala-Leu-Thr-Pro-Asp-Glu-Thr-Tyr-Val-Pro-Lys-
Leu-Phe-Thr-Phe-His-Ala-Asp-Ile-Cys-Thr-Leu-Pro-Asp-Thr-Glu-Lys-
Cys-Cys-Ala-Ala-Asp-Asp-Lys-Glu-Ala-Cys-Phe-Ala-Val-Glu-Gly-Pro-Lys-
Leu-Lys-Pro-Asp-Pro-Asn-Thr-Leu-Cys-Asp-Glu-Phe-Lys-Ala-Asp-Glu-Lys-
Glu-Ala-Cys-Phe-Ala-Val-Glu-Gly-Pro-Lys-Leu-Val-Val-Ser-Thr-Gln-Thr-Ala-Leu-Ala-
Val-His-Lys-Glu-Cys-Cys-His-Gly-Asp-Leu-Leu-Glu-Cys-Ala-Asp-Asp-Arg-
Glu-Cys-Cys-His-Gly-Asp-Leu-Leu-Glu-Cys-Ala-Asp-Asp-Arg-Ala-Asp-Leu-Ala-Lys-
Tyr-Asn-Gly-Val-Phe-Gln-Glu-Cys-Cys-Gln-Ala-Glu-Asp-Lys-Gly-Ala-Cys-Leu-Leu-Pro-Lys-
Gln-Glu-Pro-Glu-Arg-Asn-Glu-Cys-Phe-Leu-Ser-His-Lys-Asp-Asp-Ser-Pro-Asp-Leu-Pro-Lys-
Val-His-Lys-Glu-Cys-Cys-His-Gly-Asp-Leu-Leu-Glu-Cys-Ala-Asp-Asp-Arg-Ala-Asp-Leu-Ala-Lys-
Glu-Tyr-Glu-Ala-Thr-Leu-Glu-Glu-Cys-Cys-Ala-Lys-Asp-Asp-Pro-His-Ala-Cys-Tyr-Ser-Thr-Val-Phe-Asp-Lys-
ESI
MALDI
ESI & MALDI
Discussion
Analysis speed and sensitivity are the most frequently used
arguments for the proper choice of instrument configuration. For a
single sample analysis, less time is obviously needed for MDLC-
ESI than for MDLC-MALDI. This is the other way round for the
analysis of large sample amounts. However, MALDI seem to
provide significantly lower sequence coverage than ESI (using TOF
or LTQ mass analyzers) for the samples tested in this study. The
difference is bigger for high sample amounts. For laboratories
handling a large number of samples, a small fraction of each
sample may be analysed by MALDI for screening purposes, while
the greater fraction of each sample is analysed by ESI.
The optimum choice of acid for the reversed phase chromatography
eluent is different for ESI and MALDI ionization, respectively. In the
case of nano LC-ESI, formic acid (FA) is the preferable acid (Tri
Fluoro acetic acid, TFA, is frequently avoided due to reported
problems with arcing at flow rates in the nl/min range). However,
TFA is reported to provide higher chromatographic resolution than
FA. In the case of MALDI, TFA is one of the preferred acids.
Consequently, for the nano LC-MALDI interface, the optimum acid
can be used regarding both chromatographic resolution and MS
sensitivity.
From theoretical considerations one could argue that the two
ionization techniques are complementary by nature. While the
relatively high mobility of low mass peptides make them easy to
detect using ESI, these peptides are generally harder to detect
using MALDI because the peptide mass is relatively close to the
mass of the typical matrix molecules used.
References
1.Johan Axelman, Henrik Wadensten, Staffan Renlund, Anders
Tangen, Axel Parbel, Hans-Rudolf Höpker, “The use of 2DLC-
MS/MS in proteome characterization – optimization of the LC
step”, ASMS 2003, Montreal
2.Anders Tangen, Henrik Wadensten, Axel Parbel, Matthias Berg
and Hans-Rudolf Höpker, “The LC-MS Workflow: A Comparative
Study of High Throughput Nano LC and MDLC Designs”, ABRF
2004, Portland
-0.60
-0.40
-0.20
0.00
0.20
0.40
0.60
0.80
-0.60 -0.50 -0.40 -0.30 -0.20 -0.10 0.00 0.10 0.20 0.30 0.40
wc[2]
wc[1]
AA Cover
MH+
HPLC
pI
Cystein
LC-ESI-T
MALDI-TO
MDLC-ESI-LTQ vs. MDLC-MALDI-LTQ:
Improvement factor for sequence coverage using ESI-LTQ instead of MALDI-LTQ.
Sample: CAF modified tryptic peptides from six proteins purified with salt step MDLC
0
2
4
6
8
10
BS
A
Transferrin
Lysozym
e
AlcoholdehydrogenaseB-G
alactosidase
C
ytochrom
e
C
ESI/MALDISeq.Cov.
MDLC Salt Step Configuration