Centre for Protein Research, Department of Biochemistry, University of Otago
Torsten Kleffmann (Research Fellow),
Joanne Preston (Technician),
Diana Carne (Technician)
Proteomics Workflow for Gel-based and LC-coupled Mass Spectrometry
2-dimensional
gel electrophoresis
1-dimensional gel
electrophoresis
Multidimensional protein/peptide
liquid chromatography
Tryptic digestion
1-D or 2-D peptide liquid chromatography
Tryptic digestion
Analysis of
PTMs
MALDI plate spotting
MALDI ToF/ToF
LC-ESI ion trap
mass spectrometry
De novo sequencing
SEQUEST algorithm
VDLTCR
LTCR
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V P TP N V S V V D LTC R
1201
25
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V
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1387
1300
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S
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NVSVVDLTCR
V
TPNVSVVDLTCR
1102
D
L
VSVVDLTCR
CR
874
T
PNVSVVDLTCR
VVDLTCR
1009
50
SVVDLTCR
DLTCR
987
75
R e la tiv e A b u n d a n c e
Mascot search engine
TCR
Protein or peptide pre-fractionation is a
prerequisite for the reduction of sample complexity
and therefore improving the dynamic range of
protein detection and identification. Sample
complexity can be reduced by either a gel-based or
gel-free approach. The latter can include a
protein/peptide purification and pre-fractionation by
multidimensional liquid chromatography. Gel based
analyses are typically performed either by 1-D gel
electrophoresis followed by an in-gel protein
digestion and further fractionation of peptides by
liquid chromatography or by two-dimensional gel
electrophoresis.
Two-dimensional gel electrophoresis (2-DE) is
frequently used to separate and purify proteins as
single molecular species. Approximately 1500
different protein spots can be separated in a single
gel. 2-DE is a quantitative method and is used for
differentially displaying the protein complements of
different samples. However, 2-DE has some
limitations such as a low resolution of membrane
proteins and very large or very alkaline protein
species. To circumvent these limitations, proteins
can be digested with trypsin and the tryptic peptides
pre-fractionated by liquid chromatography prior to
mass spectrometry.
Liquid Chromatography (LC) of tryptic peptides is
usually performed as nano flow reversed phaseliquid chromatography (RP-LC) which is coupled
either offline or online to mass spectrometry. When
2-D chromatography is performed a strong cation
exchange chromatography is connected upstream
to RP-LC. Via an automated MALDI-plate
spotting of the collected fractions MALDI mass
spectrometry is efficiently coupled to LC
fractionation. In a 2-D LC MALDI experiment the
tryptic peptides of approximately 150-200 proteins
are resolved into ca. 400 fractions on a single
MALDI-plate which is then analysed in a tandem
time-of-flight mass spectrometer.
MALDI tandem time-of-flight mass spectrometry
combines high mass accuracy and resolution of a
TOF-analyser with highly specific precursor ion
selection for sensitive CID (collision induced
dissociation) analysis. The effective sensitivity for
MS/MS analyses is in the low fmol range.
Alternatively electrospray ionization ion trap
mass spectrometry can be coupled online to an
LC-system. However Paul Ion Trap instruments
have a low resolution whereas an efficient CID
achieves complex fragmentation patterns which are
appropriate for unambiguous protein identification
by a single peptide fragment spectrum. Thus both
instrument setups are particularly suitable for high
throughput proteomics. Such large scale analyses
generate a huge amount of data which has to be
interpreted by different software tools for
unambiguous protein identification. The spectral
data (peak lists) is searched against sequence
databases with the help of software tools like
Mascot or SEQUEST. These programs perform an
in silico digestion of the sequence database to
generate a list of peptides matching the determined
masses from the precursor ion measurements. The
fragment spectra are then scored based on the
comparison with in silico generated fragmentation
patterns of the matched database sequences.
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Not all organisms which are of great scientific interest are fully sequenced yet. Proteins of such organisms can be identified by Mascot and SEQUEST
only if they have a 100% peptide sequence match in homologous proteins of sequenced organisms. Sequence information can be gained by a manual
interpretation of fragment spectra. This procedure is referred to as de novo sequencing. However de novo sequencing is labour intensive and very
error prone. In general it provides short sequence tags which can be used for homology searches and protein identification.