Introduction in Proteomics
1
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Introduction in Proteomics
Overview
• Proteomics definition
• From genome to proteome
• Aminoacids and proteins
• What proteomics can do?
• Peptide fragmentation
• Proteomics and gel electrophoresis
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• Post translational modifications
• DD NL MS3 for phosphorylation identification
• Intact protein analysis
• Lock mass
• Protein quantification
• RP-HPLC basics
Proteins
= molecules of amino acids that perform much of life’s function
Proteome
Easy to remember
3
= set of all proteins in a cell
Proteomics
= study of protein’s structure & function
4
Proteomics represents the effort to establish the identities,
quantities, structures, and biochemical and cellular functions of all proteins in an organism, organ, or
organelle, and how these properties vary in space, time, or
Not so easy to remember
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organelle, and how these properties vary in space, time, or
physiological state.
MCP 1.10 pg 675 National Research Council
Steering committee
A true multidiscipline science
• Protein chemistry
• Mass spectrometry
• Genomics
• Bioinformatics
Proteomics
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• Bioinformatics
• Computer science
• Separation science
From Genome to
Proteome
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Proteome
From cell to gene
The genes, parts of the DNA (double helix), are the functional units of the genome.
The human genome is located right in the heart of the cells, in the nucleus.
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They hold all the information necessary to create the proteins you need.
Genes are located along thread-like
structures called chromosomes.
Protein Synthesis
Protein synthesis
is the transcriptionand translation of
specific parts of
DNA to form
proteins.
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Codons set amino acids that are used
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One Genome, multiple Proteomes
tagacgacct ggcccaacgc tgtgcccagt acaagaagga
tggctgtgac ttcgccaaat ggcgttgtgt gctcaagatc ggcaagaaca
ccccctccta ccaagctatc cttgagaatg ccaacgtact ggcacgctat
gcgtccatct gccaatccca gcgcattgtg cccattgtagagcctgaggt
gctgcctgat ggagatcacg accttgacag ggctcagaag
gtcacagaga cagttctggc cgctgtgtac aaggcactca
atgaccacca tgtcttcctg gagggcaccctcctgaagcc caacatggtg
accgcaggac agtcctgctc caagaagtac aattatgagg
acaacgctag agctacagtg ttggccctgt ccagaactgt gccagctgct
gtccctggtgtgactttctt gtcaggaggt cagtcggagg aggatgcctc
tgtcatttgg atgctatcaa caagatc
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tagacgacct ggcccaacgc tgtgcccagt acaagaagga
tggctgtgac ttcgccaaat ggcgttgtgt gctcaagatc
ggcaagaaca ccccctccta ccaagctatc cttgagaatg
ccaacgtact ggcacgctat gcgtccatct gccaatccca
gcgcattgtg cccattgtagagcctgaggt gctgcctgat
ggagatcacg accttgacag ggctcagaag gtcacagaga
cagttctggc cgctgtgtac aaggcactca atgaccacca
tgtcttcctg gagggcaccctcctgaagcc caacatggtg
accgcaggac agtcctgctc caagaagtac aattatgagg
acaacgctag agctacagtg ttggccctgt ccagaactgt
gccagctgct gtccctggtgtgactttctt gtcaggaggt
cagtcggagg aggatgcctc tgtcatttgg atgctatcaa
caagatc
The building polypeptide chain
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Central Dogma of Biology
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genome transcriptome proteome
Amino acids
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Amino acids
Amino acid
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• The human body needs 20 amino acids to be able to
make (or synthesize) its thousands of proteins.
Levels of Protein StructureAminoacid structure
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What are Proteins?
Where we find proteins?Cells and body tissues, hormones, antibodies, and enzymes.
Protein functions?
Proteins are strings of amino acids and are the active elements of cells.
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• structure providing proteins (hair and fingernails).
• help in digestion (stomach enzymes), detoxify poisons, or help fight disease.
• cell membranes proteins and are important in controlling how substances pass
through these membranes.
• enzymes proteins are catalysts for biochemical reactions.
• antibodies proteins react with foreign substances to defend the body.
Sourse of Proteins
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• Grow cells and blow them up (lyses)
• Dissect tissue sample, homogenize and lyse
• Synthesize
Sourse of Proteins ( cell fractionation)
nuclei
+ 0.25M NaClnuclei
+ 0.25M NaCl
cells
+hypotonic buffer
+0.5% NP40
cells
+hypotonic buffer
+0.5% NP40
cytosolic proteins
nucleosolic proteins cytosolic proteins
nucleosolic proteins
nuclei
+ 0.25M NaClnuclei
+ 0.25M NaCl
cells
+hypotonic buffer
+0.5% NP40
cells
+hypotonic buffer
+0.5% NP40
cytosolic proteins
nucleosolic proteins cytosolic proteins
nucleosolic proteins
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+ 0.25M NaCl
chromatin-bound Orc1chromatin-bound Orc1
nuclei
+ 0.32M NaClnuclei
+ 0.32M NaCl
pre-elution step
immunoprecipitationimmunoprecipitation
extracted nucleiextracted nuclei
+ 0.25M NaCl
chromatin-bound Orc1chromatin-bound proteins
nuclei
+ 0.32M NaClnuclei
+ 0.32M NaCl
pre-elution step
immunoprecipitationimmunoprecipitation
extracted nucleiextracted nuclei
Protein Immunoprecipitation
Immunoprecipitation (IP) is the technique of precipitating
a protein antigen out of solution using an antibody that
specifically binds to that particular protein.
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specifically binds to that particular protein.
Biological fractionation
• Necessary!! To decrease complexity before analytical
fractionation
– Abundant protein depletion
– Membrane fraction
– Soluble fraction
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– Soluble fraction
– Organellar fractionation
– Affinity chromatography
– Etc…
Separation techniques versus protein characteristics
• Charge1. Ion exchange chromatography
2. Electrophoresis
3. Isoelectric focusing
• Polarity1. Adsorption chromatography
2. Paper chromatography
3. RP chromatography
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3. RP chromatography
4. Hydrophobic interaction chromatography
• Size1. Dialysis and ultrafiltration
2. Gel electrophoresis
3. Gel filtration chromatography (size exclusion chromatography)
4. Ultracentrifugation
• Specificity1. Affinity chromatography
PI Protocol
• Lyse cells and prepare sample for immunoprecipitation.
• Pre-clear the sample by passing the sample over beads that are not coated with antibody to soak up any proteins that non-specifically bind to the beads.
• Incubate solution with antibody against the protein of interest. Antibody can be attached to solid support before this step (direct method) or after this step (indirect method). Continue the incubation to allow antibody-antigen complexes to form.
• Precipitate the complex of interest, removing it from bulk solution.
• Wash precipitated complex several times. Spin each time between washes or place tube on magnet when using superparamagnetic beads and then remove supernatant. After final wash, remove as much supernatant as possible.
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supernatant. After final wash, remove as much supernatant as possible.
• Elute proteins from solid support (using low-pH or SDS sample loading buffer).
• Analyze complexes or antigens of interest. This can be done in a variety of ways:
– SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) followed by gel staining.
– SDS-PAGE followed by: staining the gel, cutting out individual stained protein bands, and sequencing the proteins in the bands by MALDI-Mass Spectrometry
– Transfer and Western Blot using another antibody for proteins that were interacting with the antigen followed by chemiluminesent visualization.
Two amino acids can undergo a condensation reaction to form a dipeptide. Further condensation reactions result in a polypeptide.
R1-COOH + R2-NH2 R1-CO-NH-R2 + H2O
Peptide bond formation
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Amino acids are linked with the peptide bond, amide bond
The aliphatic amino acids
Glycine, Gly, G Alanine, Ala, A Valine, Val, V Leucine, Leu, L
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Glycine, Gly, G Alanine, Ala, A Valine, Val, V Leucine, Leu, L
Isoleucine, Ile, I Proline, Pro, P
The aromatic amino acids
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Phenylalanine, Phe, F Tyrosine, Tyr, Y Tryptophan, Trp, W
The sulfur containing amino acids
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Cysteine, Cys, C Methionine, Met, M
S-S bond
2Cysteines/oxidation=disulfide bond (only in extracellular and not intracellular proteins)
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Disulfide bonds stabilize protein structure by providing crosslink
The hydroxyl amino acids
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Serine, Ser, S Threonine, Thr, T
The acidic amino acids
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Aspartate, Asp, D Glutamate, Glu, E
The amide amino acids
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Asparagine, Asn, N Glutamine, Gln, Q
The basic amino acids
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Lysine, Lys, K Arginine, Arg, R
The imidazole amino acid
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Histidine, His, H
Amino acids that accept a positive charge
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Histidine
H
His
Lysine
K
Lys
Arginine
R
Arg
Aminoacids clasification
Side chains of aminoacids
Responsible for many of the
uniques properties of proteins
• charged or polar groups
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• charged or polar groups
provide interesting catalytic
groups
• nonpolar amino acids - a
protein folding issue
Amino acid 3LC SLC Average Monoisotopic
Glycine Gly G 57.0519 57.02146
Alanine Ala A 71.0788 71.03711
Serine Ser S 87.0782 87.02303
Proline Pro P 97.1167 97.05276
Valine Val V 99.1326 99.06841
Threonine Thr T 101.1051 101.04768
Cysteine Cys C 103.1388 103.00919
Leucine Leu L 113.1594 113.08406
Isoleucine Ile I 113.1594 113.08406
Monoisotopic and Average Mass
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Isoleucine Ile I 113.1594 113.08406
Asparagine Asn N 114.1038 114.04293
Aspartic acid Asp D 115.0886 115.02694
Glutamine Gln Q 128.1307 128.05858
Lysine Lys K 128.1741 128.09496
Glutamic acid Glu E 129.1155 129.04259
Methionine Met M 131.1926 131.04049
Histidine His H 137.1411 137.05891
Phenyalanine Phe F 147.1766 147.06841
Arginine Arg R 156.1875 156.10111
Tyrosine Tyr Y 163.1760 163.06333
Tryptophan Trp W 186.2132 186.07931
What Proteomics
can do?
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can do?
Types of Experiments; key questions of Proteomics
Protein separation In order identify each protein in the mixture.
Protein identificationMass spectrometry
Antibody based assays can also be used, but are unique to one sequence motif.
Edman degradation used to confirm sequence when MS unavailable.
Protein quantificationGel-based methods, differential staining of gels with fluorescent dyes (difference gel electrophoresis).
Gel-free, various tagging or chemical modification methods, label free methods.
Protein sequence analysisSearching databases.
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Searching databases.
Structural proteomics High-throughput determination of protein structures in three-dimensional space. Methods are x-ray crystallography and
NMR spectroscopy.
Interaction proteomicsThe investigation of protein interactions on the atomic, molecular and cellular levels.
Protein modification Phosphoproteomics and glycoproteomics.
Cellular proteomicsThe goal is to map location of proteins and protein-protein interactions during key cell events. Uses techniques such as
X-ray Tomography and optical fluorescence microscopy.
Methods for protein analysis
High resolution mass spectrometry methodsTop down methods
Intact proteins
Bottom up methods
Digested peptides
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Digested peptides
De Novo Sequencing
Gel based methodsOne/Two-dimensional gel electrophoresis
Protein digestion
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Why Trypsin
• Very Specific R/ K/ except R/P K/P
• Very Active
• Can buy it in a very pure form
– Promega, Sigma, Princeton Scientific
• Resistant to digesting itself
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• Resistant to digesting itself
• K and R residues in an average protein are optimally spaced
– Peptides are usually 600-3000 Da
• For electrospray, fragments are at least doubly charged because of C term basic AA’s K and R
Chemical and enzymatic cleavage reagents
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Proteomics and Mass
Spectrometry
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Spectrometry
Methods for protein analysis
High resolution mass spectrometry methodsTop down methods
Intact proteins
Bottom up methods
Digested peptides
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Digested peptides
De Novo Sequencing
Gel based methods One/Two-dimensional gel electrophoresis
Schematic representation of the top-down approach
Protein mixture
RP-HPLC
ESI-FTICR MS
25.00 50.00 75.00 100.00 125.00 150.00 175.00 200.00 225.00 250.00 275.00 300.00 325.00Time-2
100
%
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m/z
+
++
+
isolate
dissociate
+
+
+
m/z
m/z
Compare experimental
deconvoluted spectra
with predicted
spectra
Protein identification
Peptide fragments
Peptide mass fingerprintingHPLCProtein mixture
1) Denaturation2) Reduction3) Alkylation4) Trypsin digestion
Schematic representation of the bottom-up approach
Mascot
ProFound
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Mass spectrometer
Selected ion
G L F E
MS/MS
Peptide sequence tagging
Protein identification
Database
MS
m/z
m/z
ProFound
MS-Fit
Mascot
XTandem
Sequest
Mass Spectrometry based protein identification
Peptide Mass Fingerprinting (PMF)
• Determine m/z of the peptide ions only (MS)
Product Ion Scanning
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Product Ion Scanning
• Determine the m/z of the peptide ions (Parent ions)
• Fragment peptide ions
• Determine m/z of fragments (Product Ions)
Peptide Mass Fingerprinting
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Finger Print limitations
• You need a mass spectrometer capable of reasonable
accurate masses
• Genome must be pretty small
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Yeast or smaller for good results
• Mixtures of two or more proteins can be a problem
Finger Print Advantages
• Usually can give you better coverage
• Very Fast
• Easy
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• Easy
• Good preliminary screening
Product Ion Scanning
• Digest Protein with trypsin
• Determine the m/z of a peptide ion
– ESI, MALDI
• Isolate the peptide ion from any other ions
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• Fragment the peptide ion
• Determine mass of fragments
• Obtain amino acid sequence data from fragments
NR Database approx
1 Million protein
sequences
50 million tryptic
peptide sequences
SEARCHING....
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=
Match!!!
Time = 15 seconds
200 300 400 500 600 700 800 900 1000 1100 1200 1300m/z0
100
%
733.2870138
263.068777
235.076841
147.12386
270.146333 602.2563
32362.141126
271.15634
565.234920
494.195616
433.1906;7
602.758619
702.276712
733.7970105
734.296074
1104.406645734.8063
31 1033.373324
789.328120
918.347016
972.4068;6
1105.413322
1203.516213
Computer Programs
Thermo Scientific application specific software
- Xcalibur data system (operating platform, Protein
Calculator, Xtract)
- BioWorks protein identification software
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- Proteome Discoverer
- Mass Frontier (structure of compounds)
- MetWorks (drug methabolism software)
- SIEVE (differential expression software)
Search Engines
• 4 general Types
– Automated De novo sequencing
� Peaks
� Lutefisk XP
– Peptide Sequence tags
� Guten Tag
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� Guten Tag
– Cross Correlation
� SEQUEST
– Probability Based
� Mascot
� xTandem 2
� OMSSA
� PROTE_PROBE
Peptide
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Fragmentation
Peptide FragmentationRoepstorff Nomenclature for Possible Peptide Fragments
b1 b2 b3
c3 c2 c1
a3 a2 a1 H+
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NH2 C H
R1
C
OHN CH
R2
C
OHN CH
R3
C
OHN CH COOH
R4
y3 y2 y1
z3 z2 z1
x3 x2 x1
Fragmentation scheme of a tetrapeptide showing
the formation of b and y ions
NH2
CH NH
CH NH
CH NH
CH OH
R1
O
O
O
OR3
R4R2
H+
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NH2
CH NH
CH OH
O
OR3
R4
NH2
CHC
NH
CH
R1
O R2
CO
H++b2
y2
Peptide Fragmentation
(Low-Energy Collision induced fragmentation)
• Under low energy dissociation conditions, peptides primarily fragment at the C-N bond.
• If the charge is retained on the N terminal fragment, the ion is classed as either a, b or c.
• If the charge is retained on the C terminal, the ion type is either x, y or z.
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• A subscript indicates the number of residues in the fragment.
• The loss of CO from the b ion is known as a-type ion
• In addition, peaks are seen for ions which have lost ammonia (-17 Da) denoted a*, b* and y* and water (-18 Da) denoted a°, b° and y°.
The structures of the six singly charged sequence ion
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http://www.matrixscience.com/help/fragmentation_help.html
Example of y and b ions in BioWorks™
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MRFA fragmentation
61
M R F A
To better undersand
b1 b2 b3
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HCD vs. CID on MRFAE:\MRFA_CID_HCD
MRFA_CID_HCD #183-198 RT: 3.01-3.27 AV: 16 NL: 1.93E7
T: FTMS + p ESI Full ms2 [email protected] [50.00-600.00]
50 100 150 200 250 300 350 400 450 500
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
271.12236
R=84953
C 11 H 19 O 2 N 4 S
0.12432 ppm
104.05283
R=137349
C 4 H 10 N S
-0.18450 ppm
288.14883
R=82484
C 11 H 22 O 2 N 5 S
-0.13978 ppm
237.12335
R=91142
C 12 H 17 O 3 N 2
-0.09154 ppm
376.19787
R=72159
C 18 H 26 O 4 N 5
-0.16048 ppm
524.26458
R=61277
C 23 H 38 O 5 N 7 S
-0.72984 ppm
453.22758
R=67571
C 20 H 33 O 4 N 6 S
-0.60473 ppm
56.04941
R=187947
C 3 H 6 N
-1.20621 ppm
185.10318
R=103452
C 7 H 13 O 2 N 4
-0.67809 ppm
149.07424
R=118738
C 5 H 13 O N 2 S
-0.45283 ppm
454.23074
R=63433
121.08394
R=122565
HCD
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50 100 150 200 250 300 350 400 450 500
m/z
MRFA_CID_HCD #133-152 RT: 2.18-2.49 AV: 20 NL: 3.56E7
T: FTMS + p ESI Full ms2 [email protected] [140.00-600.00]
50 100 150 200 250 300 350 400 450 500
m/z
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
271.12230
R=84435
C 11 H 19 O 2 N 4 S
-0.07701 ppm
453.22767
R=65916
C 20 H 33 O 4 N 6 S
-0.38973 ppm
376.19793
R=71240
C 18 H 26 O 4 N 5
0.00283 ppm
489.22757
R=62061
C 23 H 33 O 4 N 6 S
-0.57564 ppm
418.19077
R=67476
C 20 H 28 O 3 N 5 S
0.07745 ppm
229.10048
R=89238
C 10 H 17 O 2 N 2 S
-0.18034 ppm
306.15940R=79572
C 11 H 24 O 3 N 5 S
-0.13604 ppm
181.09710
R=104712
C 9 H 13 O 2 N 2
-0.29719 ppm
153.10215
R=115156
C 8 H 13 O N 2
-0.60042 ppm
CID
0
20
40
60
80
100
Rela
tive A
bundance
524.26473R=124753
C23 H38 O5 N7 S
-0.44024 ppm
525.26739R=120897
C2213C H38 O5 N7 S
-1.77174 ppm 526.26062R=124362
C23 H38 O5 N734S
-0.27554 ppm
NL:1.17E8
MRFA_CID_HCD#252-259 RT: 4.34-4.53 AV: 8 T: FTMS + p ESI SIM ms [514.30-534.30]
Elemental composition on MRFA
measured
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524 525 526 527 528
m/z
0
20
40
60
80
100
0524.26496R=123889
C23 H38 O5 N7 S
0.00001 ppm
525.26769R=120637
C2213C H38 O5 N7 S
-1.19110 ppm 526.26101R=118269
C23 H38 O5 N734S
0.47332 ppm
NL:1.67E4
C23 H38 O5 N7 S: C23 H38 O5 N7 S1
p (gss, s /p:40) Chrg 1R: 124000 Res.Pwr . @FWHM
simulated
0
1
2
3
4
5
6
Re
lativ
e A
bun
da
nce
526.26062R=124362
C 23 H 38 O 5 N 734S
-0.27554 ppm526.27045R=130297
C2113C 2 H 38 O 5 N 7 S
-2.32164 ppm
NL:1.17E8
MRFA_CID_HCD#252-259 RT: 4.34-4.53 AV: 8 T: FTMS + p ESI SIM ms [514.30-534.30]
NL:
Isotopic distribution of 526.26 on MRFA
measured
65
526.25 526.26 526.27 526.28 526.29
m/z
0
1
2
3
4
5
6526.26101R=118269
C 23 H 38 O 5 N 734S
0.47332 ppm526.27075R=96228
C 2113C 2 H 38 O 5 N 7 S
-1.75871 ppm
NL:1.67E4
C 23 H 38 O 5 N 7 S: C 23 H 38 O 5 N 7 S 1
p (gss, s /p:40) Chrg 1R: 124000 Res .Pwr . @FWHM
simulated
Isotopic contribution of 13C and 34S in MRFA
66
Detection Time & Mass Resolution
526.26112
526.27090
526.26112
526.27090
13C2 and 34S isotopes
C23H38N7O534S 526.2607 u 13C2C21H38N7O5S 526.2716 u
∆m = 0.0109 RP (m/∆m) = 48,000
524.0 525.0 526.0 527.0
m/z
0
50
100
Rela
tive A
bundance
524.26489
525.26761
526.26112527.26415
MRFAMolecular ion region
67
526.20 526.25 526.30 526.35
m/z
0
10
20
30
40
50
60
70
80
90
100
Rela
tive A
bundance
526.26551
Detect time: 375 ms; RP: 28,000
Detect time: 750 ms; RP: 55,000
Detect time: 1500 ms; RP: 95,000
„Freedom of fragmentation“ (and detection)
• Combination of three different and complementary
fragmentation techniques CID, HCD, and ETD for sequence
assignments with absolute confidence.
• Most comprehensive solution for
– complex PTM analysis
68
– complex PTM analysis
– intelligent sequencing of peptides
– top-down and middle-down analysis
– protein quantitation via stable isotope labelling such as
iTRAQ™ or label-free quantitation
[M + 2H]+•
[M + 2H]2+ + A- [M + 2H]+• + A
Electron Transfer Dissociation (ETD)
[c+H]+ + [z+H]+•
c z••••
69
c z
5
10
15
20
25
30
35
40
Re
lative A
bu
nd
an
ce
1216.75003
1094.416691347.50003899.58335624.58335496.50001
751.41668
856.66669552.50001424.41667 1003.75002 1172.75003
NL: 9.46E4
SubP_SA#1 RT: 4137.28 AV: 1 T: ITMS + p ESI sid=35.00 sa Full ms2 [email protected] [185.00-2000.00] with supplemental activation
Unreacted precursor ions
c4 c5 c6 c7c8
c9c10
Importance of supplemental activation for ETD
70
400 500 600 700 800 900 1000 1100 1200 1300
m/z
0
5
10
15
20
25
30
35
40
0856.66669552.50001424.41667 1003.75002 1172.75003
1364.58337
1216.75003
1103.66669 1319.50003899.66669
752.58335 1171.66670624.58335 1001.66669854.66669496.58334
NL: 1.13E5
SubP_noSA#1 RT: 4136.94 AV: 1 T: ITMS + p ESI sid=35.00 Full ms2 [email protected] [185.00-2000.00]
without supplemental activation
Charge-reduced
species
c4c5
c6c7
c8 c9
c10
Unreacted precursor ions
Example: Substance P
• ETD has no low mass cut off and provides for a complete interpretation of the spectrum.
• ETD enables sequencing of larger peptides.
• Peptide sequence information from CID and ETD spectra are complementary.
ETD vs CID
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• CID/ETD toggle method improves sequence coverage and increases protein ID confidence.
• With ETD, high quality spectra are obtained for >2+ precursor ions.
• Supplemental activation improves ETD of 2+ ions.
• Digestion with LysC, AspN and GluC may provide additional benefits for proteome analysis with ETD.
CID and HCD of 667.74852+ (Sulfatation)
200 300 400 500 600 700 800 900 1000 1100 1200 1300
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
627.76886z=2
CID
NL: 79.9593qSDDyGHMRF-amid
72
100 200 300 400 500 600 700 800 900 1000 1100 1200 1300
m/z
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
646.32288z=1 809.38684
z=1
452.24274z=1
428.14029z=1
589.30151z=1
924.41412z=1
1039.44141z=1
321.20282z=1
136.07515z=1 537.20648
z=2
271.12192z=1 1126.47327
z=1
HCD
m/z
0
2 0
4 0
6 0
8 0
1 0 0
Rela
tive
Ab
und
an
ce
4 9 6 . 7 9 1 6 9
7 7 2 . 4 8 2 3 6
2 3 9 . 1 1 3 5 3 7 0 1 . 4 4 4 8 9 8 7 3 . 5 3 0 3 3
3 8 4 . 2 7 1 2 1
HCD vs CID HTAGFIPRL-amide
CID
73
2 0 0 4 0 0 6 0 0 8 0 0 1 0 0 0
m / z
0
2 0
4 0
6 0
8 0
1 0 0
01 1 0 . 0 7 0 6 9
7 7 2 . 4 8 2 0 6
8 8 3 . 5 1 4 5 3
7 0 1 . 4 4 4 6 4
4 9 6 . 7 9 1 5 63 6 7 . 2 4 4 4 8
6 0 9 . 3 1 3 1 7
HCDHimm
HCD MS/MS Spectrum of Perisulfakinin
74
Methods for protein analysis
High resolution mass spectrometry methodsTop-down methods
Intact proteins
Bottom-up methods
Digested peptides
75
Digested peptides
De Novo Sequencing
Gel based methods One/Two-dimensional gel electrophoresis
Automated de novo Sequencing of
DPGWNNLKGLWamide using PEAKS™ Software
76
Problems !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
• You have two ladders of overlapping masses
• Cannot tell which is a b ion or which is a y ion
• Incomplete fragmentation
• Chemical noise
• Mass accuracy of some instruments are not good
77
• Mass accuracy of some instruments are not good
enough to determine R groups– Glutamine and lysine differ by 0.036 .
– Phenylalanine and oxidized methionine differ by 0.033
– Gly + Val = 156.090 u and Arg = 156.101
Big Problem
• Can take you a very long time to “sequence” a “good”
product ion spectra without a computer
– 30 minutes if your good
– 1-2 days to never if you are not
78
– 1-2 days to never if you are not
• One experiment can generate 10,000 MS/MS spectra
• Proteins due to their nature are very complex
eg. cysteine – cysteine cross-linking, tertiary structure
formation and post-translational modifications)
Beside the amount...
79
• Proteins can be very large in size and hard to handle.
Decrease complexity
• Separate proteins (before digestion)– 2-d electrophoresis– 1-d electrophoresis– Chromatography (RP, cation/anion exchange, size excision)– Immunoafinity– Molecular weight-centrifugation, ultrafiltration, solvent precipitation
• Separate peptides (after digestion)– Multidimensional chromatography (MUDPIT)
80
– Multidimensional chromatography (MUDPIT)
Reducing agents – detergents (SDS, CHAPS), salts (urea, guandine), acids (formic)
Break sulfur bridges – DTT and use iodoacetamide to keep them from reforming
Heat
Proteomics and Gel
81
Electrophoresis
Methods for protein analysis
High resolution mass spectrometry methodsTop down methods
Intact proteins
Bottom up methods
Digested peptides
82
Digested peptides
De Novo Sequencing
Gel based methodsOne/Two-dimensional gel electrophoresis
2-D electrophoresis
2-DE is a powerful separation technique, which
allows simultaneous resolution of thousands of
proteins.
83
2-D electrophoresis
Disadvantages– Modest detection limit
– High abundance proteins
– Protein bias (pI)
– Difficult to automate
– Labor intensive
– Requires many more mass
84
– Requires many more mass spec runs
Advantages– High resolution separation
– Can get quantitation by staining
– Good “snapshot” of the Proteome!
Approach used to identify a spot from a 2-D gel analysis
Excision ofprotein spot
2-D gel
Peptide fragments
Mass spectrometer
Peptide mass fingerprinting
Gel spot
DigestionExtraction
MS
85
spectrometer
Selected ion
G L F E
MS/MS
Peptide sequence taggingSearch
Protein identification
Database(NR, EST)Database(NR, EST)
Search
1-D electrophoresis
• Disadvantages– Difficult to automate
– Very limited expression profiling
• Advantages– No staining necessary
In gel trypsin digestion
[65
-78
]
[57
7-5
96
]
M IS S W1 W2 W3 E1 E2 E3
94
67
45
M IS S W1 W2 W3 E1 E2 E3
94
67
45
A
In gel trypsin digestion
[65
-78
]
[57
7-5
96
]
[65
-78
]
[57
7-5
96
]
M IS S W1 W2 W3 E1 E2 E3
94
67
45
M IS S W1 W2 W3 E1 E2 E3
94
67
45
A
86
– No staining necessary
– High protein recovery
– Decreased runs needed
• 2-50 proteins can be identified per band or section
1200 1700 2200 2700 m/z
[20
8-2
15
][6
16
-623
][2
35
-242
][7
49
-756
]
[38
-46
]
[75
7-7
67
]
[47
-55
]
[42
0-4
30
][3
92
-403
][4
7-5
7]
[27
1-2
83
]
[33
7-3
51
]
[46
4-4
78
]
[37
2-3
91
]
[47
9-4
95
]
[18
-35
]
[19
5-2
15
]
[47
9-5
03
]
B
1200 1700 2200 2700 m/z
[20
8-2
15
][6
16
-623
][2
35
-242
][7
49
-756
]
[38
-46
]
[75
7-7
67
]
[47
-55
]
[42
0-4
30
][3
92
-403
][4
7-5
7]
[27
1-2
83
]
[33
7-3
51
]
[46
4-4
78
]
[37
2-3
91
]
[47
9-4
95
]
[18
-35
]
[19
5-2
15
]
[47
9-5
03
]
1200 1700 2200 2700 m/z
[20
8-2
15
][6
16
-623
][2
35
-242
][7
49
-756
]
[38
-46
]
[75
7-7
67
]
[47
-55
]
[42
0-4
30
][3
92
-403
][4
7-5
7]
[27
1-2
83
]
[33
7-3
51
]
[46
4-4
78
]
[37
2-3
91
]
[47
9-4
95
]
[18
-35
]
[19
5-2
15
]
[47
9-5
03
]
B
Typical MUDPIT Preparation
(Multidimensional Protein Identification Technology)
Protein digest mixtures
SCX fractionation
CID
Micro
electrospray
ionization
RPfractionation
• Advantages
– Dynamic Range 105
– Sensitivity! Low
abundance proteins
– Minimized protein bias
– Highly automated
87
MS-1 MS-2
Auto MS/MS detection
Tandem Mass Spectra
Database searchprotein I.D.
• Disadvantages
– Poor isoform &
modification distinction
– Still overwhelms the
mass spectrometer
– Does not give you a
very good “snapshot”
Post Translational
Modification
88
Modification
After Translation
Post-translational modifications
• Phosphorylation – Ser, Thr, Tyr
• Methylation – Lys
• Oxidation – Met
89
• Oxidation – Met
• Deamidation – Asn, Gln
• Glycosylation
PTM Scanning
• Can search data for unexpected modifications from the
unimod database
www.unimod.org
90
Ways to look for PTM’s
• Mass spectrometry– Good for PTM’s with a stable mass
• Phosphorylation
• Methylation
• Acetylation
– Difficult to analyze large modifications
• Large carbohydrates
91
• Large carbohydrates
• Edman Sequencing• Phosphorylation
• Radio-labeling– Very sensitive
– Usually hard to get the identity or AA with PTM
The Trinity of Protein Phosphorylation Analysis
II. Identify the sites of phosphorylation
I. Identify the kinase responsible for phosphorylation
� Predict potential phosphorylation sites with the Scansite program
(http://scansite.mit.edu)
� Gel kinase assay
92
II. Identify the sites of phosphorylation
III. Identify the function of phosphorylation
� Generate mutants forms of protein for in vitro testing
� Separate phosphoproteins by SDS-PAGE
� Digest in-gel the phosphoproteins to generate (phospho)peptides
� MS/MS analysis
Most important part of finding PTM’s
• Protein Coverage!
93
Problems with Mass spec approach
• Cannot prove a negative
– Just because you do not see your PTM does not
mean it is not there!
94
Some common PTM’s
95From Jensen et al. nature march 2003 vol 21
Data Dependent NL MS3 method setup for
96
method setup for phopshorylation
identification
LTQ Orbitrap MS LTQ Orbitrap MS
Instrument configuration settings
97
Configuration window: important settings
98
Data Dependent NL MS3
99
NL in MS2 trigger MS3
100
Scan events
1. First scan
event is the
reference scan
(usually a FTMS
full scan)
2. All subsequent
scans events may
be dependent on
scan event 1.
101
scan event 1.
3. A scan
depending on a MS
scan will produce a
MS/MS spectrum. A
scan depending on
a MS/MS scan will
produce a MS3
spectrum
Scan event 2
102
Scan event 2. Settings
103
NL masses
104
Scan event 2 determined from Scan event 1
.
105
Scan event 3 (MS3 of the peaks which lost phosphate)
106
Monoisotopic precursor selection toggle…
see next slide
107
… And What It Does
• Problem: monoisotope signal is lower abundant than first isotope⇒ wrong precursor ion mass would be used for db searches ⇒ wrong results
• Monoisotope Toggle reads the
108
• Monoisotope Toggle reads the charge state…
• …calculates a theoretical pattern for the measured m/z…
• …determines the correct monoisotope signal ⇒ correct results from db search
Non-Peptide Monoisotopic Recognition…
see next slide
109
…And What It Does
• determines a charge state (isotope distance) for a
detected signal cluster
• looks for a signal left from the most abundant signal
• this signal needs to have at least 60% intensity of the
110
• this signal needs to have at least 60% intensity of the
highest one
• the toggle will only work if the isotope cluster follows a
‚standard intensity deviation‘ → ‚Cl‘ or ‚Br‘ containing
cluster will not work
New features
• Use m/z values as Mass
– Triggers only one charge state out of a CS distribution
• Chromatography Trigger
– Peak apex triggering
111
– Peak apex triggering
• NL Triggered HCD
– Performs HCD fragmentation on precursors, which carry modifications, which result in CID spectra with NL
“Use m/z values as Mass”
112
� triggers only the most intense charge state of a charge state distribution
“Use m/z values as Mass”
60
70
80
90
100
Rela
tive A
bu
nd
an
ce702.11792
z=4
561.89581z=5
539.08704z=5
113
400 500 600 700 800 900 1000
m/z
0
10
20
30
40
50
Rela
tive A
bu
nd
an
ce
673.85785z=4 722.35474
z=3
935.82166z=3803.94409
z=2388.22092z=2
897.80676z=3
Neutral Loss Triggered HCD
OT-FS
OT-CID1
OT-CID2
NL?OT-HCDno
yes
114
OT-CID2
OT-CID3
OT-HCD
OT-HCD
NL?
NL?
RT: 0.00000 - 39.96544
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38
Time (min)
0
20
40
60
80
100
Re
lative
Ab
un
da
nce
30.99966478.31247
31.07783478.31296
11.53153589.80402
13.32670495.75748
31.23676478.31235
15.00021717.86932
28.56122353.1505717.43568
678.8215310.94827
410.19772 31.38696478.31326
24.80037499.22260
21.24148367.12732
2.21873311.98303
39.76065519.13251
4.55575519.13116
36.70453519.13214
NL: 7.07E7
Base Peak F: FTMS + p NSI Full ms [300.00-1400.00] MS ft051117_DEK_16_MDM_TBB_K25_ph
ft051117_DEK_16_MDM_TBB_K25_ph #746 RT: 12.53 AV: 1 NL: 1.40E6T: FTMS + p NSI Full ms [300.00-1400.00]
50
100
Re
lative
Ab
un
da
nce
633.36344R=65778
z=3475.27383R=88272
z=4
802.42220R=51415
z=11097.15152
R=32148z=3
707.28542R=57530
z=3
380.22460R=136210
z=5
944.39175R=44574
z=2
533.26548R=101970
1144.00233R=29975
z=?
984.36977R=35330
z=?
852.89723R=34052
z=?
x10
FTMS
Base Peak Chromatogram
*-1.02 ppm
Identification of phosphorylated Ser-303 in DEK
protein using NLMS3 method
115
300 400 500 600 700 800 900 1000 1100 1200 1300 1400
m/z
0
Re
lative
Ab
un
da
nce
z=3z=3
R=136210z=5
z=2R=101970
z=2 z=?R=35330
z=?R=34052
z=?
ft051117_DEK_16_MDM_TBB_K25_ph #750 RT: 12.57 AV: 1 NL: 4.70E3T: ITMS + c NSI d Full ms2 [email protected] [250.00-1900.00]
300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800
m/z
0
20
40
60
80
100
Re
lative
Ab
un
da
nce
895.4
886.5
926.5 1289.1599.2470.2 1418.1877.4 1191.11003.2714.2 822.4581.2 1118.2 1741.31630.31532.4433.1
967.2
823.7
345.2309.0
ft051117_DEK_16_MDM_TBB_K25_ph #751 RT: 12.58 AV: 1 NL: 1.39E2T: ITMS + c NSI d Full ms3 [email protected] [email protected] [235.00-1805.00]
300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800
m/z
0
20
40
60
80
100
Re
lative
Ab
un
da
nce
1191.1886.4
1320.2599.2 877.4
470.1 714.2 787.1 1192.21076.2822.4 1003.2581.31643.21532.21321.31173.2765.6
1118.3
1302.41004.3 1661.4985.3700.6
600.1
474.1 1626.31514.5
916.3
1445.4373.2943.2
327.3 1334.4
1463.1
529.7
407.0
ITMS2 (944.39)
ITMS3 (895.43)
299KESEpSEDSSDDEPLIK314
299KESEDhaEDSSDDEPLIK314
[M-H3PO4+2H]2+
299KESEpSEDSSDDEPLIK314
pm -H3PO4
pm -H3PO4 -H2O
Identification of phosphorylated Ser-303 in DEK protein
using NLMS3 method and Bioworks
IT MS2
116
299KESEDhaEDSSDDEPLIK314
pm -H2O
pm -2H2O
IT MS3
Identification of the phosphorylation site Ser-39 from
eIF3c subunit using neutral loss/MS3 method
117
Multiphosphorylation of the N-terminal part of eIF3c
118
Neutral Loss Scanning
119
(A) elimination of phosphate from phosphothreonine to produce dehydroamino-2-butyric acid (scheme isalso valid for phosphoserine where dehydroalanine forms) and phosphoryc acid
(B) absence of the same mechanistic pathway for b-elimination of phosphate from phosphotyrosine.
Mechter_1880_3+_ETD_FT_Recal #1-20 RT: 0.01-0.31 AV: 20 NL: 3.18E5T: FTMS + p NSI Full ms2 [email protected] [120.00-1500.00]
55
60
65
70
75
80
85
90
95
100
Re
lative A
bu
nd
an
ce
459.2254z=3
LTQ Orbitrap XL ETD for Phosphopeptide Analysis
RpSRGGKLGpSLGK
[M+3H]3+
[M+3H]2+•
120
200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400
m/z
0
5
10
15
20
25
30
35
40
45
50
55
Re
lative A
bu
nd
an
ce
623.7946z=2
1076.4759z=1
688.8367z=2
554.2560z=1
1360.6494z=1
341.1339z=1
908.4700z=1798.3886
z=1 1036.5426z=1
739.3722z=1497.2346
z=1
301.2004z=1
1247.5928z=1
1343.6226z=1
1150.5386z=1
258.1456z=?
426.5608z=3
938.5614z=1
∆pS
∆pS
[M+3H]2+•
174.1356
z=1
Phosphopeptide standards courtesy of Karl Mechtler, IMP Vienna, Austria
LTQ Orbitrap XL ETD for Phosphopeptide Analysis
using ETD
121Phosphopeptide standards courtesy of Karl Mechtler, IMP Vienna, Austria
CID and HCD of 667.74852+ (Sulfatation)
200 300 400 500 600 700 800 900 1000 1100 1200 1300
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
627.76886z=2
CID
NL: 79.9593qSDDyGHMRF-amid
122
100 200 300 400 500 600 700 800 900 1000 1100 1200 1300
m/z
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
646.32288z=1 809.38684
z=1
452.24274z=1
428.14029z=1
589.30151z=1
924.41412z=1
1039.44141z=1
321.20282z=1
136.07515z=1 537.20648
z=2
271.12192z=1 1126.47327
z=1
HCD
m/z
Intact protein
analysis
123
analysis
C1080 H1697 N268 O325 P5 S6
Beta-casein (5x phosphorylated)
Analysis of intact phosphoproteins reveals the
number of phosphorylation sites
124
experimentalsimulated∆∆∆∆m = 1.3 ppm
RELEELNVPGEIVESLSSSEESITRINKKIEKFQSEEQQQ
TEDELQDKIHPFAQTQSLVYPFPGPIPNSLPQNIPPLTQT
PVVVPPFLQPEVMGVSKVKEAMAPKHKEMPFPKYPVEPFT
ESQSLTLTDVENLHLPLPLLQSWMHQPHQPLPPTVMFPPQ
SVLSLSQSKVLPVPQKAVPYPQRDMPIQAFLLYQEPVLGP
VRGPFPIIV
1
209
- 5 HPO3
Alkaline Phosphatase
Dephosphorylation by AP treatment reveals the number of
phosphorylation sites
125
Amino acid sequence of bovine Carbonic Anhydrase II
SHHWGYGKHNGPEHWHKDFPIANGERQSPVDIDTKAVVQDPALKPLALVY
GEATSRRMVNNGHSFNVEYDDSQDKAVLKDGPLTGTYRLVQFHFHWGSSD
DQGSEHTVDRKKYAAELHLVHWNTKYGDFGTAAQQPDGLAVVGVFLKVGD
ANPALQKVLDALDSIKTKGKSTDFPNFDPGSLLPNVLDYWTYPGSLTTPP
LLESVTWIVLKEPISVSSQQMLKFRTLNFNAEGEPELLMLANWRPAQPLK
Ac-
126
LLESVTWIVLKEPISVSSQQMLKFRTLNFNAEGEPELLMLANWRPAQPLK
NRQVRGFPK
Sum Formula: C1312H1996N358O384S3
Monoisot. Mass: 29,006.682670
N-Terminus: N-Acetylation
Full FTMS of the Bovine Carbonic Anhydrase II
700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400
m/z
0
50
100
Rela
tive A
bundance
937.25580R=80000
854.64618R=84404
1001.82526R=80500807.22192
R=843001076.03418
R=71501 1161.95667R=70104
1210.32898R=73100
745.25647R=91604
1320.17651R=65704 1383.28064
R=65304
0
50
100
Rela
tive A
bundance
937.25580R=80000
907.99872R=78304
968.46460R=76400
1001.82526R=80500
880.54437R=77500
951.14423R=82400
1007.26550R=83300
127
∆∆∆∆m = 0.44 ppm
880 900 920 940 960 980 1000
m/z
0
Rela
tive A
bundance
28955 28960 28965 28970 28975 28980 28985 28990 28995 29000 29005 29010
m/z
0
50
100
Rela
tive A
bundance
29006.66975
29010 29015 29020 29025 29030 29035
m/z
0
50
100
Rela
tive A
bundance
29023.7186329026.72841
29020.7163529028.73130
29018.71561 29030.72314
29032.7259629016.71637
CAIII_SIM_1001 #1 RT: 38.6 AV: 1 NL: 1.21E6T: FTMS + p ESI Full ms3 [email protected] [email protected] [275.00-1400.00]
0
20
40
60
80
100
Rela
tive A
bundance
1001.85901R=79200
1033.56897R=63604
951.12561R=67704
436.66791R=108704
866.92560R=39704
1155.01184R=48704
1320.69739R=45804
527.01331R=88404
337.77481R=104904
673.80261R=72301
Isolation of the [M+29H]29+ ion of the Bovine Carbonic
Anhydrase II
128
300 400 500 600 700 800 900 1000 1100 1200 1300 1400
m/z
CAIII_SIM_1001 #1 RT: 38.6 AV: 1 NL: 1.21E6T: FTMS + p ESI Full ms3 [email protected] [email protected] [275.00-1400.00]
1001.0 1001.5 1002.0 1002.5 1003.0 1003.5
m/z
0
20
40
60
80
100
Rela
tive A
bundance
1001.85901R=79200
1001.96216R=78904
1001.68750R=76604
1002.03101R=77304
1001.61847R=77104 1002.10010
R=75904
1002.54681R=70604
1003.20111R=75104
1002.20349R=69404
1002.75409R=71704
1001.51563R=724041001.24005
R=67204
1003.44348R=755041000.99670
R=67204
Higher Energy Collision Dissociation (HCD) of the [M+34H]34+ ion
T: FTMS + p ESI Full ms2 [email protected] [100.00-1400.00]
18
20
22
24
26
28
30
Re
lative
Ab
un
da
nce
539.28272z=1
159.09247z=1 519.04469
HCD (854.0)
129
200 400 600 800 1000 1200 1400
m/z
0
2
4
6
8
10
12
14
16
Re
lative
Ab
un
da
nce
z=1 519.04469z=5 836.80323
z=?
337.18795z=1
HCD Fragmentation Details using ProSight PTM
130
HCD Fragmentation Details using ProSight PTM
131
T: FTMS + p ESI Full ms2 [email protected] [235.00-1400.00]
60
70
80
90
100
Re
lative
Ab
un
da
nce
951.14404z=8
592.93689z=5
807.23987z=19
1095.21057z=8
CID (854.0)
Collision Induced Dissociation (CID) of the [M+34H]34+ ion
132
400 600 800 1000 1200 1400
m/z
0
10
20
30
40
50
Re
lative
Ab
un
da
nce
1251.52588z=7
1134.52502z=1
CID Fragmentation Details using ProSight PTM
133
CID Fragmentation Details using ProSight PTM
134
Ubiquitin_ETD_11 #1-106 RT: 0.01-6.13 AV: 106 NL: 1.10E3T: FTMS + p ESI Full ms2 [email protected] [210.00-2000.00]
55
60
65
70
75
80
85
90
95
100
Re
lative A
bu
nd
an
ce
277.1328z=1
948.1839z=3
1136.6501z=1
390.2168z=1 869.7391
z=4
ETD spectrum of Ubiquitin, 12+ charge state, with
Orbitrap detection
*
135
400 600 800 1000 1200 1400 1600 1800
m/z
0
5
10
15
20
25
30
35
40
45
50
55
Re
lative A
bu
nd
an
ce
z=4
537.2850z=1
1347.2301z=2790.4653
z=2636.3535
z=11012.2238
z=3764.4484
z=1
1108.7627z=6
1159.3174z=3
1273.6870z=4
669.3921z=2 1421.7778
z=6433.2516z=2
1702.7334z=5
1486.8083z=5
305.1328z=?1
* http://upload.wikimedia.org/wikipedia/commons/a/ac/Ubiquitin_cartoon.png
FUNDAMENTAL: Mass Accuracy
Parts per million = [Masstheor - Massexp ]
Massx 106
(PPM)
136
Masstheor(PPM)
Avandia_pos_05a #670 RT: 17.24 AV: 1 NL: 8.27E6F: FTMS + p ESI Full ms [100.00-1000.00]
55
60
65
70
75
80
85
90
95
100
Rela
tive
Abundance
358.1217
Mass Accuracy and Resolving Power
N NO
NH
S
O
O
Rosiglitazone
C18H19N3O3S
[M+H]+ 358.12199
error: 0.77 ppm
R 60,000 @m/z400
⇒⇒⇒⇒ R = 77,000
137
358.2 358.4 358.6 358.8 359.0 359.2 359.4 359.6 359.8 360.0 360.2
m/z
0
5
10
15
20
25
30
35
40
45
50
55
Rela
tive
Abundance
359.1249
360.1175
Mass Accuracy and Resolution: Clozapine
Clozapine_Mix #2104 RT: 25.47 AV: 1 NL: 2.03E5T: FTMS + p NSI Full ms [90.00-800.00]
326.5 327.0 327.5 328.0 328.5 329.0 329.5
m/z
0
10
20
30
40
50
60
70
80
90
100
Re
lative A
bu
nd
an
ce
327.1374R=9300
329.1340
R=9300328.1407
R=9200
Clozapine_Mix #813 RT: 2.93 AV: 1 NL: 4.15E5T: FTMS + p NSI Full ms [150.00-800.00]
326.5 327.0 327.5 328.0 328.5 329.0 329.5
m/z
0
10
20
30
40
50
60
70
80
90
100
Re
lative A
bu
nd
an
ce
327.1371R=18400
329.1338R=18800
328.1406R=18600
R 7,500 Scan 0.3 sec R 15,000 Scan 0.3 sec
error: 0.9 ppm error: <0.1 ppm
138
Clozapine_Mix #849 RT: 3.27 AV: 1 NL: 2.15E5T: FTMS + p NSI Full ms [150.00-800.00]
326.5 327.0 327.5 328.0 328.5 329.0 329.5
m/z
0
10
20
30
40
50
60
70
80
90
100
Re
lative A
bu
nd
an
ce
327.1369R=74500
329.1340R=74600
328.1402R=73600
Clozapine_Mix #875 RT: 3.93 AV: 1 NL: 1.65E5T: FTMS + p NSI Full ms [150.00-800.00]
326.5 327.0 327.5 328.0 328.5 329.0 329.5
m/z
0
10
20
30
40
50
60
70
80
90
100
Re
lative A
bu
nd
an
ce
327.1368
R=147700
329.1339R=146300
328.1402R=148900
R 60,000 Scan 1 sec R 100,000 Scan 1.8 sec
error: 0.6 ppmerror: 0.9 ppm
Different scan speeds and resolution – Mass accuracy < 1ppm
Mass spectrometers do not measure mass, but m/z!
� If a peptide with a mass of 700 Da gets ionized and
acquires 2 protons at pH 3.0
� The mass spectrometer sees
700+2 = 702/2 = 351.00
Mass to charge (m/z)
139
700+2 = 702/2 = 351.00
� This mass is referred to as [M+2H]2+
100
60
80
648.82
649.32
Determining Charge State
649.32-648.82=0.5
1/0.5=2 (charge state)
140
648 649 650 651
m/z
0
20
40
649.81
650.31
(648.82*2)-2 = M 1295.64
m/z= 648.82
Lock Mass
141
Lock Mass
Ions which can be used as lock masses during
online LC-MS analyses
The
polydimethylcyclosiloxane
(PCM) ions generated in the
electrospray process from
ambient air (protonated
(Si(CH3)2O))6; m/z 445.120025) were used for
internal recalibration in real
142Olsen, Mann et al. Mol. Cell. Proteomics 2005, 4: 2010-2021
“Parts per million mass accuracy on an orbitrap mass
spectrometer via lock-mass injection into a C-trap.”
internal recalibration in real
time.
Detection
Internal calibration = Lock Mass
Injection of the calibrantInjection of analyte
Mixing of ion populations and ejection
143
Olsen, Mann et al. Mol. Cell. Proteomics 2005, 4: 2010-
2021
“Parts per million mass accuracy on an orbitrap mass
spectrometer via lock-mass injection into a C-trap.”
Internal Calibration
• Lock masses are isolated in the LTQ with a reduced
inject time.
• The C-Trap is filled with them. The reduced inject time
adjusts the lock mass intensity to appr. 5%.
• The LTQ is filled for a second time using the full inject
time.
144
time.
• Ions from the second filling are moved to the C-trap and
mixed with the previous ion population of the calibrant.
• Ions are shot into the orbitrap.
• After the transient acquisition and data processing an
existing calibration function is adjusted based on the
measured frequencies of the lock masses.
Getting the most from your lock mass
• For a specific compound of interest use a lock mass
close in mass
• Note: if you have a lock mass at low m/z but your
compound has high m/z, it might be better to use
external calibration
• Generally: we recommend using 1 lock mass only
145
• Generally: we recommend using 1 lock mass only
• If you put more lock masses, you need fill time for each
of them, and it does not necessarily improve the result
• If no lock mass is found the system applies the last
external calibration
• Thus, you should keep your orbitrap externally well
calibrated anyway!
Internal Calibration: Tune Setup
146
1. Locking ON
2. Enter accurate mass of a known compound
Internal Calibration: Method Setup
• Enable lock mass in the current method ‘L’
• Can edit lock masses
• A lock mass from the ‘method’ take preference over the
lock mass from ‘Tune’ file
147
Ionisation and desorption in ESI mass spectrometry
Electrospray Solvated
Macromolecular Ion
Aerosol droplets Desolvated
Macromolecular Ion
spray
Electrospray Solvated
Macromolecular Ion
Aerosol droplets Desolvated
Macromolecular Ion
spray
148
Ions are formed by spraying the solution from a fine needle into an electric field at
atmospheric pressure.
The droplets pick up charges, in the form of protons; solvent evaporates as the
droplets enter the lower pressure regions of the instrument leaving “naked” intact
peptides with varying numbers of charges
(z =1,2,3,4…n).
Protein
Quantification
149
Quantification
Why Quantitate on the protein level
• There can be a big disconnect between the mRNA level
and the protein level
– Protein Turn over
– Post-translational modifications
150
• Gel based
• LC-MS based
• Label free approaches
• Stable isotope labelling
Quantitation in Proteomics
151
• Stable isotope labelling
Differential proteomics
Comparison of the relative amounts of proteins between
two or more biological samples
- Diseased vs healthy
- Larva vs pupa vs adult
152
- Larva vs pupa vs adult
- Sepsis - die vs live
• Samples to be run in tandem
• Labeled and unlabeled peptides to elute together (LC)
• Labeled and unlabeled peptides ionize similarly (ESI)
Labeling methods
Allow to:
153
• Labeled and unlabeled peptides ionize similarly (ESI)
Quantitation with Isotope Labels
• Incorporate a ‘mass tag’ into protein or peptide
– Create heavy and light version
• Mix the labeled samples
• Analyse together
• Use mass spectrometer to differentiate the heavy from
light versions
154
• Compare to get the ratio
Light (L)
1036 1037 1040 1043 1044 10450
10
20
30
40
50
60
70
80
90
100
Rela
tive
Abundance
1039.5125z=2
1043.0231z=2
1038 1039
1038.5087z=2
1039.0102z=2
1038.0074z=2
Heavy (H)
1041 1042m/z
1041.0171z=2
1042.0200z=2
1042.5215z=2
1041.5186z=2
Ratio
H:L = 2.1
General idea: Differential proteomics
Enzymatic digestion (Trypsin)
LC/MS/MS
In the LC, heavy and light co-elute
MS
155m/z
rela
tive a
bundance
Normal (12C)
Abnormal (13C)
Peptide found to be
up regulated in
abnormal sample
MS/MS
rela
tive a
bundance
MS/MS
Fragment ion masses
help to identify
peptide sequence
belonging to a
specific protein
General idea: Differential proteomics
fragment ions
156
m/z
rela
tive a
bundance
specific protein
Protein determined to
be up regulated in
abnormal sample
Stable isotope labeling methods
- Isotope-Coded Affinity Tagging (ICAT)
- Hydrogen vs deuterium (8 Da separation)
- 13C vs 12C (9 Da separation)
- Isotope Tagging for Relative and Absolute protein Quantitation (iTRAQ)
- Four isobaric reagents
157
- Four isobaric reagents
- Compare quantity of reporter molecule in MS/MS
- Stable Isotope Labeling of Amino acids in Cell culture (SILAC)
-Cell growth in stable isotope-enriched media (13C Glucose, 15N Ammonium, 2H Water)
Isotope-Coded Affinity Tagging (ICAT)
X = Hydrogen (Light)
or
Deuterium (Heavy)
8 Da difference
158
http://dir.niehs.nih.gov/proteomics/emerg2.htm
Binds to and modifies
cysteine residues
(alkylation)
Used to affinity
capture reacted
cysteine containing
peptides (avidin)
ICAT
- Analyze only the peptides containing cysteine
- Reduce complexity of sample
- Cys relative abundance 2.5%
- Problems with getting the hydrogen and deuterium labeled -
- peptides to co-elute
159
- Changed linker chain so that carbons were isotopically
labeled (13C vs 12C, 9 Da separation)
- 13C and 12C peptides co-elute
- Reducing complexity also means missing a lot of peptides
and reduction in confidence in protein identification
Stable Isotope Labeling of Amino acids in Cell
culture (SILAC)
� SILAC uses in vivo metabolic incorporation of “heavy” 13C- or 15N-labeled amino acids into proteins followed by mass spectrometry-based analysis.
� ‘Kits’ commercially available
160
Everly, P.A., et al. (2004).Quantitative cancer proteomics: Stable isotope labeling with amino acids (SILAC) as a tool for prostate cancer research. Mol & Cell Proteomics. 3.7: 729-735.
Mann, M. (2006). Functional and quantitative proteomics using SILAC. Nature Reviews. 7:952-959.
SILAC workflow
161Mann, M. (2006). Functional and quantitative proteomics using SILAC. Nature Reviews. 7:952-959.
Isotope Tagging for Relative and Absolute protein
Quantitation (iTraq)
162
iTraq is a registered Trademark od Applied Biosystems
iTraq-Chemistry
163
covalently links an iTRAQ™ reagent
isobaric tag with each lysine side chain
and N-terminal group of a peptide
ensures that an iTRAQ™ reagent-labeled peptide,
whether labeled with iTRAQ™reagent 114, 115,
116, or 117, displays at the same mass
RT: 0.00000 - 47.89528
60
65
70
75
80
85
90
95
100
Rela
tive A
bundance
10.06990721.39038
21.85394778.72766
16.06159854.79303
26.23417883.45465
6.41333446.94089
Base peak chromatogram and MASCOT identification
of proteins from the PRG study sample
*
******
*
1640 5 10 15 20 25 30 35 40 45
Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
Rela
tive A
bundance
446.94089
11.21418677.84949
5.96203317.22058
16.67878855.12280
30.468841204.97388
5.68283384.24384
40.22073432.23828
34.65009404.207492.31799
323.16766
C:\Documents and Settings\...\ft050930_5 9/30/2005 3:43:18 PM itraq_1
RT: 0.00000 - 47.89528
0 5 10 15 20 25 30 35 40 45
Time (min)
0
50
100
0
50
100
Re
lative
Ab
un
da
nce 10.06990
721.3903821.85394
778.7276616.06159854.79303
26.23417883.45465
6.41333446.94089 30.46884
1204.97388 40.22073432.23828
34.65009404.20749
45.88895308.22272
2.31799323.16766
21.42729844.45465
NL: 2.83E7
Base Peak F: FTMS + p NSI Full ms [300.00-1600.00] MS ft050930_5
NL: 7.87E6
m/z= 844.44786-844.46474 F: FTMS + p NSI Full ms [300.00-1600.00] MS ft050930_5
ft050930_5 #2288 RT: 21.43 AV: 1 NL: 7.72E6F: FTMS + p NSI Full ms [300.00-1600.00]
50
100
Re
lative
Ab
un
da
nce
844.45465R=49220
z=2563.30560R=74662
z=3896.09943R=47783
z=31132.62744 1522.14624672.07874 1388.82874831.95197 1001.13818545.85303 1206.24377436.46356380.55933
MS2 identification of an iTRAQ labeled tryptic peptide from BSA
and quantification of the tag in a subsequent MS3 scan
FTMS
*
165
300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600
m/z
0
Re
lative
Ab
un
da
nce
z=31132.62744
R=35651z=?
1522.14624R=26857
z=?
672.07874R=71282
z=4
1388.82874R=28179
z=?
831.95197R=57961
z=2
1001.13818R=38096
z=?
545.85303R=70488
z=?
1206.24377R=28228
z=?
436.46356R=78460
z=?
380.55933R=61628
z=?
ft050930_5 #2268 RT: 21.27 AV: 1 NL: 3.68E4T: ITMS + c NSI d Full ms2 [email protected] [220.00-1700.00]
300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700
m/z
0
50
100
Re
lative
Ab
un
da
nce
965.2723.3866.3291.1
822.4652.2 1083.41036.3719.3605.2 1397.5966.2
1183.3724.3 867.2406.2 1212.4 1282.31084.4 1543.5505.3 969.4821.6653.2345.1 1442.5868.3466.1 534.2 1545.51085.5 1526.51019.4 1214.5246.1 761.3 1661.4
ft050930_5 #2272 RT: 21.30 AV: 1 NL: 4.62E3T: ITMS + c NSI d Full ms3 [email protected] [email protected] [70.00-595.00]
100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290
m/z
0
50
100
Re
lative
Ab
un
da
nce
246.1
116.9 274.1113.9 144.9
230.2163.0
228.0147.0 273.1100.8
569TVMENFVAFVDK580
ITMS2
ITMS3
FTMS
y1
MS/MS identification of the iTRAQ labeled peptide 569TVMENFVAFVDK580
166
Precursor Ion 1319.144 (2+)
HCD MS/MS of iTRAQ labelled Peptide
167
50
60
70
80
90
100
Re
lativ
e A
bu
nd
an
ce
Carbonic Anhydrase (1 : 0.5 : 0.2 : 0.03)
10
20
30
40
50
60
70
80
90
100
Rela
tive
Ab
und
an
ce
114.11012
115.10724
116.11040
117.11372
# bSeq
.y #
1 244.1778 V 9
2 357.2618 L 1018.5901 8
3 472.2888 D 905.5060 7
4 543.3259 A 790.4791 6
168
200 400 600 800 1000 1200
m/z
0
10
20
30
40
50
Re
lativ
e A
bu
nd
an
ce
114 115 116 117
m/z
0 5 656.4099 L 719.4420 5
6 771.4369 D 606.3579 4
7 858.4689 S 491.3310 3
8 971.5530 I 404.2989 2
9 K 291.2149 1
HCD/iTRAQ
• No „low mass cutoff“ with HCD fragmentation
• Good quantitation in the low mass region
• Good sensitivity
169
• Good sensitivity
�No limits to detect reporter ions and
fragments in one spectrum
�Good peptide ID
�Good quantitation
Label-Free Approach
Sample 1
Sample 2
170
• Align chromatographic traces (The quality of the alignment software is a key parameter in the comparative LC-MS procedure)• Match peptide mass/charge state• Quantify based on elution profile of each peptide• Identify using MS/MS spectra
171Comparative LC-MS: A landscape of peaks and valleys, Proteomics, 2008Antoine H. P. America and Jan H. G. Cordewener
ABRF Competition PRG 2007
Thermo Fisher: BRIMS (LTQ Orbitrap)
Thermo Fisher: SJ Proteomics Marketing (LTQ Orbitrap)
172
RP-HPLC basics
173
RP-HPLC basics
Analysis and purification of
proteins and peptides by Reversed-Phase HPLC
• Separation of peptide fragments from enzymatic digests.
• Purification of natural and synthetic peptides.
• Study enzyme subunits and research cell functions.
174
• Study enzyme subunits and research cell functions.
• Analysis of protein therapeutic products.
• To verify conformation and to determine degradation
products in intact proteins.
Mechanism of interaction between polypeptides and
RP-HPLC columns
175
RP-HPLC separates polypeptides based on subtle differences in
the “hydrophobic foot” of the polypeptide being separated.
Isocratic versus gradient elution
Shallow gradients can be used very effectively to separate
similar polypeptides where isocratic separation would be
impractical.
176
The role of the column in polypeptide separations
by RP-HPLC
• The HPLC column provides the hydrophobic surface onto which the polypeptides adsorb.
• Columns consist of stainless
177
• Columns consist of stainless steel tubes filled with small diameter, spherical adsorbent particles, generally composed of silica whose surface has been reacted with silane reagents to make them hydrophobic.
Adsorbent Pore DiameterPolypeptides must enter a pore in order to be adsorbed and separated.(pores of about 300 Å) Some peptides (<~2,000 MW) may also be separated on particles of
100 Å pores.
Adsorbent Particle Size
The role of the column in polypeptide separations
by RP-HPLC
178
Smaller diameter particles generally produce sharper peaks and better resolution.
Adsorbent Phase TypeHydrophobic phase is usually a linear aliphatic hydrocarbon of eighteen
(C18), eight (C8) or four (C4) carbons.
Column Dimensions: Length
• Column length does not significantly affect separation and resolution of proteins.
• Consequently, short columns of 5–15 cm length are often used for protein separations.
179
often used for protein separations.
• Columns of 15–25 cm length are often used for the separation of synthetic and natural peptides and enzymatic digest maps.
• Column back-pressure is directly proportional to the column
length.
Column back-pressure
180
• In case of viscous solvents, such as isopropanol, shorter
columns will result in more moderate back-pressures.
Column dimensions: diameter
When the diameter of an HPLC column is reduced
1. flow rate is decreased
2. solvent used decreased
3. detection sensitivity is increased (smaller amounts
181
3. detection sensitivity is increased (smaller amounts of polypeptide can be detected).
Very small diameter HPLC columns ((75 µm Diameter)
are particularly useful when coupling HPLC with mass spectrometry (LC/MS).
Columns, example
182
Organic modifier
The organic modifiers solubilizes and desorbs the polypeptide from the hydrophobic surface.
183
Organic solvents used in LC/MS
Acetonitrile (ACN)
■ It is volatile and easily removed from collected fractions.
■ It has a low viscosity, minimizing column back-pressure.
■ It has little UV adsorption at low wavelengths.
184
■ It has little UV adsorption at low wavelengths.
■ It has a long history of proven reliability in RP-HPLC
polypeptide separations.
Isopropanol
■ Used for large or very hydrophobic proteins.
disadvantage of isopropanol is its high viscosity.
Organic solvents used in LC/MS
185
■ To reduce the viscosity of isopropanol - use a mixture of 50:50 acetonitrile: isopropanol. Adding 1–3% isopropanol to acetonitrile has been shown to increase protein recovery in some cases.
Ethanol
■ Elute hydrophobic, membrane-spanning proteins and is
used in process purifications.
Methanol
Organic solvents used in LC/MS
186
Methanol
Dichloromethane
Clorofprm
Hexane
The ion-pairing reagents or buffers sets the eluent pH and interacts with the polypeptide to enhance the separation.
Ion-pairing reagents and buffers
187
Ion-pairing reagents and buffers
Trifluoroacetic acid
■ It is volatile and easily removed from collected fractions.
■ It has little UV adsorption at low wavelengths.
■ It has a long history of proven reliability in RP-HPLC
188
■ It has a long history of proven reliability in RP-HPLC
polypeptide separations.
■ Enhancement of chromatographyc resolution.
■ Adverse effect on ions formation in LC/MS
Heptafluorobutyric acid (HFBA)
■ Is effective in separating basic proteins.
Triethylamine phosphate (TEAP)
■ Has been used for preparative separations.
Ion-pairing reagents and buffers
189
Formic acid (FA)
■ In concentrations of 10 to 60%, has been used for the chromatography of very hydrophobic polypeptides.
Ion exchange chromatography orthogonal analytical
techniques
The benefits of ion exchange chromatography
■ high sample loading capacity.
■ crude solutions can be loaded
The benefits of Reversed Phase chromatography
■ a high degree of selectivity based on differences in hydrophobicity or molecular conformation.
190
■ crude solutions can be loaded onto ion-exchange columns.
■ addition of urea, acetonitrile or
non-ionic detergents to break-up
complexes.
■ optimization of elution selectivity
■ use of volatile buffers or ion-pairing agents.
■ freedom from interferences by
salt or buffers from ion exchange.
LC/MS additives and buffers summary
Protons donors
Acetic acid
Formic acid
Proton acceptors
Amonium hidroxid
Amonia solutions
191
Ion-Pair reagents
Trichloroaceticacid (low than 0.02% v/v)
Trifloroaceticacid
Triethylamine
Trimethylamine
Buffers
Amonium acetate
Amonium formate
How to make a gradient
192
193