Transcript mass

Proteomics I
Mass Spectrometry
please study
Functional Genomics by Mass Spectrometry
(Andersen and Mann, 2000)
FEBS Letters 480, 25-31
Proteomics II
for Monday
Yeast Two Hybrid
please study
Toward a Protein-Protein Map of the Budding
Yeast
(Ito et al., 2000)
PNAS 97(3), 1143-1147
Mass Spectrometry
• Molecules to be analyzed, referred to as analytes are first
ionized (usually in a vacuum),
• Newly charged molecules are introduced into an electric
and/or magnetic field in gas phase,
• Their path through the field is a function of the mass to
charge ratio m/z,
• m/z of the ionized species can be used to deduce the mass
of the analyte with high precision.
Biological Samples
....bringing polypeptides and nucleic acids to the gas
phase usually degrades the molecules,
1988
matrix assisted laser desorption/ionization mass spectrometry
MALDI-MS
electrospray ionization mass spectrometry
ESI-MS
Proteases
...proteins are first degraded into smaller
peptides by sequence specific proteases,
– assists in elution from gels and other sources,
– large polypeptides give “indefinite” masses.
MALDI-MS
...peptides are suspended in a matrix of lightabsorbing molecules,
– deposited onto a solid substrate,
• high-voltage is applied to the solid substrate,
– laser excitation of the matrix,
• peptides are “released” from the matrix, and accelerate through
the electrical field,
• ionized occurs during desorption.
MALDI-MS
E = 1/2 mv2
Ionization is Variable
peak-to-peak difference
1 proton
Cipherin’
(m/z)1
=
M + (n2 + 1)X
n2 + 1
(m/z)2
=
(m/z) = mass/charge ratio
M = mass of peptide
n2 = number of charges
X = mass of protons
M + n2X
n2
Two Formulas, Two Unknowns
(m/z)1
=
M + (n2 + 1)X
n2 + 1
(m/z)2
=
M + n2X
...solve for n2,
n2
=
(m/z)1 - X
(m/z)2 - (m/z)1
...then solve for M,
M = n2[(m/z)2 - X]
n2
Multiple Computations
Each protein yields
multiple peptides, with
highly resolvable
masses.
MALDI Peptide Mass Mapping
“Mass Fingerprinting”
...proteins are cleaved in a sequence specific manner,
– thus, each protein in a proteome has a unique peptide
mass subset,
• these subsets can be computationally derived from protein
databases, and translated genomic DNA sequences,
• experimentally determined unknowns can be compared, via
computers, to online databases for identification,
..scalable, multiple samples can be deposited at
once, computers sort out the constituents.
Figure 1a.
mass
MALDI MS Mass Fingerprinting.
However
...protein databases are not yet inclusive,
– protein fingerprint data is not available, or is
inconclussive for large parts of most genomes,
...some proteins are too small to give “enough”
peptide fragments for fingerprinting,
...computer deconvolution has it’s limits.
Electrospray Ionization Mass Spectrometry
ESI-MS
• Peptides analytes, in solution, are passed through a charged
needle that is kept at high electrical potential,
–
–
–
–
the peptides are ionized,
this disperses the the solution into a fine spray,
the solvent quickly evaporates,
peptides now in gas phase,
• Enter mass spectrometer for mass fingerprinting,
or
Peptide Sequencing.
ESI-MS
Figure 1b.
mass
Mass Spectrometry via Electro-Spray Ionization (ESI-MS).
Tandem Mass Spectroscopy
(MS-MS)
Often provides
enough information
to unambiguously
identify the entire
protein in protein, or
translated genomic
databases.
...mass spectrometry can also be used to obtain
sequence to identify peptides,
– treatment with sequence specific proteases provides
information of the terminal residues,
– the mass of the entire peptide is determined,
– a short amino acid sequence from the peptide.
b-type ions (a-carboxyl)
y”-type ions (a-amino)
Figure 1c.
693.37(EYL)1098.55
...single entry in the
database,
+
total peptide mass
=
TQLYEYLQR
MALDI Dual Quadrupole
MALDI MS-MS
Combines MALDI-MS scalability with ESI-MS sequencing.
Genome Searching
...we now have the ability to match heterologous MS
data to ‘raw’ genomic data,
– i.e. unannotated, untranslated DNA sequence from the
genome projects,
– i.e. don’t need “complete” protein sequences for
fingerprinting.
Multi-protein Complexes
?
...i.e. nuclear pore complexes,
...i.e. cellulose synthase complexes,
...i.e. spindle pole apparati,
...i.e. proteins involved in the spliceosome, etc.
...optional reading, available online...
Isolate Spliceosome Complex
Anti-m3G Antibody Chromotography
Rather than co-precipitating with an anti-m3G antibody and separating
on a gel, use the anti-m3G antibody to purify the complex using
chromotography.
Histidine Tag Snp1
Nickel Nitrilotriacetic Acid (Ni-NTA) Affinity Chromotography
...Snp1 has been identified as a U1 specific
snRNP,
...the functional Snp1, in the experiment
(yeast), is carried on a plasmid,
...it has been modified by a hexahistidine
addition to the N-terminus,
...the exposed his-tag binds to lead on a
chromatography column, is washed off
with imidazole.
Separate Ni-NTA Eluate
... SDS-PAGE fractionation yields 20 proteins,
• bands are in-gel digested,
• extracted,
• send to MS-MS for ID.
Identify U1 Specific Bands
Glycerol Centrifugation to
Separate after Ni-NTA.
SDS Page Comparisons
MS-MS
1. fragment at 756.9
was selected for
sequencing,
2. LEEL sequenced,
3. AELEELEEFEFK
unique match in
the database.
Other partially sequenced
peptides correspond
(underlined).
Whole Complex De-convolution
* Prp39p (106 aa peptide)
• Prp40p (41 aa peptide)
...peptides can be isolated and from heterogenous
samples, sequenced and ID’d.
Found Players
• All of the known U1-specific proteins found to date were
identified,
• An ortholog to human U1-C was found that had not been
identified in yeast was detected,
• Four novel proteins were also identified.
Establishment of Principle
...so, in one fell swoop, years of molecular genetic and
biochemical research was replicated,
and
...a U1-C protein, not detected above, was identified as a
component in the system,
and
...four novel proteins have been ID’d,
– not necessarily components,
– but excellent research leads.
Use of Principle
...in one subsequent experiment with isolated human spliceosome,
– 70 spliceosome protein spots were analyzed,
– 19 novel splicing factors were identified,
and
...all 19 could be cloned directly via EST libraries,
and
...in vivo conformation of the role of these factors in splicing was
obtained through co-localization studies using green fluorescence
protein (GFP).
Quantitation
...progress has been made in using MS to
quantify protein expression,
– requires labeling of one or more protein species
and is generally limited to relative expression.
Signaling Pathways
(4.2)
Organelle
(4.3)
please study
Functional Genomics by Mass Spectrometry
(Andersen and Mann, 2000)
FEBS Letters 480, 25-31
Neubauer et al. available online or reference.