MASS SPECTROMETRY IN PROTEOMICS
The advantages of identifying proteins via mass spectrometry as
compared to traditional methods include:
•
High sensitivity (femtomole to low picomole range),
allows analysis of Coomassie- and silver-stained proteins.
•
High specificity provides unique protein identification.
•
Mass determination of several peptides from a protein
results in verification of a significant part of the protein
sequence.
•
Identification of several proteins in a single sample can be
done; many samples can be processed in parallel.
•
If the protein is identified as "unknown", the sequences
obtained by mass spectrometry can be used to query EST
and genomic databases. The identified nucleotide
sequences can then be used for cloning the gene.
Outline of mass spectrometric analysis of
proteins from SDS gels
Digestion
A few percent
MALDI Peptide Map
To identify proteins from 1-D and 2-D gels, the
protein spots are excised and placed in 96-well
plates. The gel plugs are washed, and cystine
residues are reduced and alkylated.
The protein is cleaved with trypsin and the peptides
generated are eluted from the gel piece. A small
aliquot is analyzed by MALDI mass spectrometry
and the masses of the tryptic peptides are
determined.
The digested sample is purified by chromatography on a
microcolumn packed in the tip of a glass capillary. The
sample is eluted into 0.3-1 mL, and analyzed by nanoelectrospray tandem mass spectrometry.
Digestion
In ESI, a strong electrical charge is imparted to the solvent
Containing the protein
Outline of mass spectrometric analysis of
proteins from SDS gels
1. Ionization
MALDI (Matrix-Assisted Laser
desorption/ionisation
ESI (electrospray ionization)
2. Mass spectrometer (separation based on
mass/charge of the protein, m/z ratio)
3. Activation
4. Mass spectrometer (Mass determination)
Outline of TOF-TOF analysis
In TOF-TOF analysis, the separation in MS1 is used to select ions of
a specific mass. These are then broken up either by smashing into
neutral gas molecules (“collision induced dissociation”) or by further
laser irradiation. The mixture of fragments is then subjected to mass
analysis (MS2).
Outline of MALDI mass spectrometric analysis
Protein or peptide sample is spotted on a metal plate, being
co-crystallized with “matrix”, typically 3,5-dimethoxy-4hydroxycinnamic acid (sinapinic acid), α-cyano-4hydroxycinnamic acid (alpha-cyano or alpha-matrix) or 2,5dihydroxybenzoic acid (DHB).
The matrix absorbs light from a UV laser, and co-volatalizes
with the sample, which then enters the mass spectrometer.
Outline of mass spectrometric analysis
Sample is inserted into the mass
spectrometer. A vacuum is drawn.
The laser is fired and the resultant
charged ions are accelerated by a
fixed high voltage electric field.
Larger ions move slower than smaller
ions, and so their time-of-flight (TOF)
defines their masses.
This is an example of a sector MS, in which the mass differences are
detected by analyzing the deviation of the ions in a magnetic field.
MS instruments can have a linear detection arrangement, or a
reflective mode (“reflectron”), in which the ions are caused to follow a
parabolic path back to the detector – which increases the path length
and hence the mass accuracy.
Outline of mass spectrometric analysis of
proteins from SDS gels
1. Ionization
MALDI (Matrix-Assisted Laser
desorption/ionisation
ESI (electrospray ionization)
2. Mass spectrometer (separation)
3. Activation
4. Mass spectrometer (Mass determination)
Activation step during Mass Spectrometry
After selection by m/z ratio, proteins are
broken into smaller fragments.
Send proteins through argon-filled chamber
Collision with gas molecules and the
vibration energy generated causes the
proteins to break into two pieces (b and y
pieces, the amino and carboxyl pieces
respectively)
Breaks can occur anywhere in the protein
•
This is fed into the second MS unit, which is operated
under conditions that lead to partial fragmentation of
the peptide. The resulting masses are measured.
•
The mass values are then compared to those
theoretically obtained from fragmentation of known
peptide sequences.
•
The high mass accuracy and high resolution in the
mass spectra allow very confident sequence
assignments.
•
To query sequence databases, the peptide mass and
sequence are combined into a Peptide Sequence
Tag. This results in a very high search specificity.
•
Unique hits in the database can be obtained with
only 3-4 amino acids.
•
When a protein has been identified, its theoretical
fragmentation is predicted and compared to the
MS/MS spectrum for assignment of other peaks. This
allows validation of the identification.
•
This procedure is repeated for every fragmented
peptide in the sample and leads to additional
verification of the result or identification of other
proteins in the sample.
Matching proteins in the database query are scored and
ranked by the number of matching peptide masses, mass
accuracy and protein size.
In many cases, as shown above, you can unambiguously
identify the protein. A large number of different peptides
within the protein sequence correspond to the MALDI-MS
mass values.
Some protein samples cannot be identified by the MALDIMS peptide masses.
•
The number of peptide masses may be too low for a
specific database query.
•
The gel piece may contain a mixture of proteins.
•
The protein is novel and the sequence is not present
in the database.
In this case, more specific database queries can be
performed for some of the peptides using partial sequence
information derived by MS analysis.
•
Note: You can use the protein sequence information
to produce oligonucleotide sequences that can be
compared to available DNA sequence databases.
Since each protein produces several peptide
sequences, this provides redundancy and allows
confidence in identification of genes.
ICAT Technology
Gygi SP, Rist B, Gerber SA, Turecek F, Gelb
MH, Aebersold R (1999).
Quantitative analysis of complex protein
mixtures using Isotope-Coded Affinity
Tags. Nature Biotechnology 17:994-999.
•
The main difficulty of applying original methods of mass
spectrometry to mixtures of proteins (as found within
cells) is that the methods were not capable of monitoring
changes in concentrations of specific proteins.
•
A second problem is that these methods are biased
towards highly abundant proteins, and lower abundance
regulatory proteins are seldom detected when total cell
lysates are analyzed.
•
Isotope-Coded Affinity Tag (ICAT) technology addresses
both of these problems.
ICAT technology involves:
•
A pair of chemical reagents that react with sulfhydryl
groups in proteins, that can specifically add mass, and that
provide a means for selective purification of the modified
peptides.
•
One of the pair of reagents contains deuterium, and this
means that the same peptide modified by the two
different reagents can be distinguished using MS.
•
Prior to MS analysis, the treated protein samples are
subjected to proteolysis and the modified peptides are
isolated by affinity chromatography.
LCMS
MS is used here to quantify
the different modified
peptide-pairs.
MSMS
Tandem MS is used here to
identify the individual
modified peptides.
ICAT technology works because:
•
A short sequence of contiguous amino acids (5-25 long) is
sufficient to uniquely identify a protein.
•
Pairs of peptides tagged with the light and heavy ICAT
reagents are chemically identical, and there provide
excellent mutual internal standards for quantification.
•
Measurement of the ratios of the upper and lower mass
components of the peaks of pairs provides a very accurate
measurement of the relative abundances of the peptides
and hence the proteins.
Test of the method
•
Six proteins were selected, and two mixtures of the
proteins were made, having different but known
compositions.
•
The two mixtures were separately labeled with the ICAT
reagents. They were then mixed, treated with protease,
the tagged peptides affinity purified, and separated using
HPLC.
•
Each protein produced several peptides. These were
analyzed using MS (to determine the proportion of the two
mass isoforms) and MS-MS to get the sequences of the
individual peptides.
MS analysis of material eluting at one time point from the HPLC run.
Four mass doublets are identified. The proportions of each can be
measured easily. MS-MS sequencing of the peptide at a m/z ratio of
998 is shown in the next figure.
MS-MS analysis of the m/z = 998 peptide.
Note the process has high redundancy (multiple peptides for specific
proteins). Also tagging and enrichment for cysteine-containing
peptides greatly reduces the complexity of the analysis.
Second test of the method
•
Used yeast growing either in 2% galactose, or in 2%
ethanol.
•
Glucose repression causes minimal expression of large
numbers of proteins with metabolic functions required for
growth on other carbon sources. This effect is reversed
by growth in the absence of glucose in the presence of
ethanol or galactose.
•
They isolated and labeled 100 mg soluble protein from each.
2% of the sample was analyzed.
•
Sequence information was collected for more than 800
different peptides.
•
Table 2 shows results for selected genes (34 in all).
Second test of the method
•
This illustrates 10 genes identified as
being glucose repressed and 21 other
genes for comparison.
•
Genes known to be expressed during
growth on galactose (GAL1, GAL10) or
ethanol (ADH2, ACH1) were detected
and quantified.
•
Multiple peptides were found for
some proteins (i.e. PCK1 produced
five, and they gave a mean ratio of
1.57 ± 0.15 (95% confidence), with an
error of <10%). Changes are in
agreement with literature and with
changes in staining intensities on
SDS-PAGE.
•ICAT method can perform analyses that other
methods (such as DNA microarrays) cannot
•Isozymes of Alcohol dehydrogenases (ADH1 and
ADH2) are 88% identical in the coding region, yet
could not be distinguished by DNA microarrays.
•The two forms are 93% identical at the amino acid
level, yet could be distinguished by ICAT method that ADH2 is present at 200 times more in ethanol
compared to galactose growth
The case of ADH1 and ADH2
•
Expression of the alcohol dehydrogenase family of
isoenzymes allows yeast growth on hexoses (ADH1) or
ethanol (ADH2).
•
ADH2 is both glucose and galactose repressed. It converts
ethanol to acetaldehyde.
•
In the presence of sugar, ADH1 converts acetaldehyde to
ethanol.
•
Regulation of these isoenzymes is one of the keys to
carbon utilization in yeast.
•
These isoenzymes differ only by a single valine to threonine
change. Can they be separated and quantified by the ICAT
method?
The case of ADH1 and ADH2
•
Yes! The detected ICAT
peptides differ in HPLC
retention time by >2 min,
and the mass by 2 Da.
•
ADH1 was expressed
about 2-fold higher when
galactose was the carbon
source, compared to
ethanol. Ethanol induction
of ADH2 was a 200-fold
increase compared to
galactose induction.
Summary
•
The ICAT method is an ideal basis for the automated,
quantitative global proteome analysis.
•
It reduces the complexity of the peptide mixture since it
relies on selective analysis of cysteine-containing peptides.
•
•
A theoretical tryptic digest of the entire yeast
proteome (6,113 proteins) would produce 344,855
peptides. Only 30,619 contain cysteine.
•
This reduces complexity but maintains
quantification.
The ICAT coupling reaction can be done in SDS and urea
(i.e. the proteins are maintained in a denatured state, and
so are not likely to degrade).
Summary
•
Avidin affinity chromatography is particularly well suited
for the HPLC-MS procedures, since it allows one-step,
compatible clean up of the digested, coupled sample
peptides.
•
The purification step eliminates many abundant
components, so increases the sensitivity.
•
The method can be redundant, ie. reproducible.
•
The method is accurate and has a reasonable dynamic range
(up to 100-fold changes can be measured).
•
Different specificities of ICAT reagents can be envisaged
for proteins that lack cysteines.
Han DK, Eng J, Zhou H, Aebersold R
Quantitative profiling of differentiationinduced microsomal proteins using IsotopeCoded Affinity Tags and mass spectrometry.
Nature Biotechnology 19:946-951 (2001).
•
Interested in looking at the protein components of
microsomal membranes.
•
Classical protein separation methods don’t work well with
membrane proteins; it’s particularly hard to keep the
proteins soluble during the isoelectric focusing step.
•
Applied ICAT techniques to HL-60 microsomal membrane
proteins to look at cellular differentiation.
•
Note that ICAT technique does not involve 2D-gel step.
This allowed the analysis of membrane proteins.
MS methods for analysis of Protein
Phosphorylation
A systemic approach to the analysis of protein
phosphorylation
Zhou H, Watts JD, Aebersold R
Nature Biotechnology 19:375-378 (2001)
•
Reversible protein phosphorylation controls a widerange of biological functions.
•
Determination of the sites of protein
phosphorylation is important.
•
Previous methods require purification of individual
proteins – very slow and laborious.
•
Need rapid and general method for identification of
protein phosphorylation in complex mixtures.
•
This is a description of such a method, using MS.