Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
Outline
• Liquid Chromatography• Electrospray• MS Acquisition • Peptide Fragmentation• PTM Detection• Protein ESI-MS
Terminology
• ESI• Liquid chromatography• Acquisition• DDA (data dependent
acquisition)• MS1 vs MS2• Precursor ion• MS/MS• Product Ions• b-ion, y-ion
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
Gingras A et al, 2007 Nat Rev Mol Cell Biol 8, 645
A. Obtain protein-complexB. Separate proteins on 1D gelC. Excise protein bands > trypsin digestionD. Liquid Chromatography-ESI-Mass Spectrometry
SDS-PAGE
EXAMPLE Workflow:MIXTURE OF UNKNOWN PROTEINS
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
Liquid Chromatography (LC): Peptide Separation
Electrospray Ionization (ESI)
Mass Spectrometry (MS)
Example Sample Analysis Workflow
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
Liquid Chromatography: Separation Technique
Mixtures are separated or partially separated before MS analysis with Chromatographic Technologies
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
http://www.chemistry.adelaide.edu.au/external/soc-rel/content/lc-col.htm
“… modern Liquid Chromatography (LC), uses a liquid mobile phase to transportthe sample components through a column packed with a solid material - thestationary phase.” Reference: http://www.earl2learn.com
From Tswett’s notebook (1910) on theearly chromatographic experiments:plant pigments were passed throughcalcium carbonate using petroleumether
Mikhail Tswett (1872 – 1919)
Liquid Chromatography Basic Overview
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
i022_091711_sPRG_pep_digestmix_HC... 9/17/2011 11:27:17 PM
0.00 - 70.01
0 5 10 15 20 25 30 35 40 45 50 55 60 65
0
10
20
30
40
50
60
70
80
90
100
41.21
36.2726.1338.809.43
31.80
24.56
33.72
19.7028.01 48.65
18.35 20.4944.71
30.98
17.65 44.87
55.2515.6450.56 57.679.06
63.0352.05 58.728.53 67.1212.712.57
• Peaks represent analyte elution profiles
• Increased Retention Time = increased peptide hydrophobicity on a C18 column
Liquid Chromatography, Peptide Elution Profile 1D LC
Time (minutes)
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
Minutes
10 20 30 40 50 60 70
• Column 1 Elution Profile• Use fraction collector – collect peptides in separate tubes
Column 2: LC-MS Peptide Elution Profiles
Mas
s Spe
c Ab
solu
te In
tens
ity
2 minute intervals
Time (sec)
Inte
nsity
, mA
U(2
14 n
m)
Liquid Chromatography, Peptide Elution Profiles 2D LC
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
Liquid Chromatography (LC): Peptide Separation
Electrospray Ionization (ESI)
Mass Spectrometry (MS)
Example Sample Analysis Workflow
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
Electrospray Ionization (ESI)Produce Analyte Ions
GOAL: eliminate solvent, get analyte into the gas phase; apply high voltage to a liquid to create aerosol
http://www.newobjective.com/electrospray/index.html
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
Electrospray Ionization
Ref: IJAC Vol 2012 ID 282574
Mas
s Spe
ctro
met
er
Liquid flow
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
Liquid Chromatography (LC): Peptide Separation
Electrospray Ionization (ESI)
Mass Spectrometry (MS)
Example Sample Analysis Workflow
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
Typical Numbers of Proteins Identified:
• 1D LC-MS: up to 300 • 2D LCMS: up to 5000
(sample and dynamic range dependent)
EXAMPLE Workflow:MIXTURE OF UNKNOWN PROTEINS
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
EXAMPLE:MIXTURE OF UNKNOWN PROTEINS
Data-Dependent Acquisition Scan Mode (DDA) on the Mass Spectrometer
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
RT: 0.67 - 73.58
5 10 15 20 25 30 35 40 45 50 55 60 65 70Time (min)
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
39.08
28.5642.32 46.09 53.64
49.6159.65
24.4532.33 54.1934.17
57.26
17.92 62.4422.91 63.7868.3717.6713.80
1.63 4.17
Next slide: see Full Scan/Survey Scan at 39 minutes
Retention time 39 minutes
1 Dimensional LC-ESI-MS Elution Profile
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
georg_hamel_011713_12503_chymo_tubA_025dda #4335 RT: 39.22 AV: 1 NL: 5.65E7F: FTMS + c NSI Full ms [360.00-1800.00]
500 600 700 800 900 1000 1100 1200 1300 1400m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
851.95
782.40671.68
587.05553.61829.91
504.01
1173.091007.02739.37616.32 929.73
689.84
1136.261063.51
863.44
1239.31 1350.02 1427.47445.12
666.34
477.43
Mass Spectrum at 39.22 minutes shows co-eluting peptides
MS1 Data Acquisition• MS1 spectrum (below)• Peaks below represent unfragmented peptide m/z values
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
georg_hamel_011713_12503_chymo_tubA_025dda #4335 RT: 39.22 AV: 1 NL: 5.65E7F: FTMS + c NSI Full ms [360.00-1800.00]
500 600 700 800 900 1000 1100 1200 1300 1400m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
851.95
782.40671.68
587.05553.61829.91
504.01
1173.091007.02739.37616.32 929.73
689.84
1136.261063.51
863.44
1239.31 1350.02 1427.47445.12
666.34
477.43
1
2345 6
Data Dependent Acquisition: • Top 6 (most abundant) Peaks Identified• Next 6 Scan Events are MS/MS
Sequential MS/MS scan events are triggered for the 6 most abundant peaks
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
MS2 Data Acquisition (1st peak triggered in DDA)MS/MS spectrumPrecursor 851.95 m/z
georg_hamel_011713_12503_chymo_tubA_025dda #4329 RT: 39.20 AV: 1 NL: 7.09E6T: FTMS + c NSI d Full ms2 [email protected] [111.00-1715.00]
200 300 400 500 600 700 800 900 1000 1100 1200 1300m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e Ab
unda
nce
279
226187159
727812
911410 628509
280
136
491 794325 826527385 709610 893 10981011956412
241
728
912
x5
peptide fragment ions peak
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
• Select Precursor Peptide• Isolate Precursor ions in Collision Cell• Fragment precursor ions with Collision
Induced Dissociation (CID)• Measure m/z of Product ions
Tandem Mass Spectrometry (MS/MS)
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
+LLLYSSQICK+
Collision CellQuadrupole Mass Filter
+ LLL+LLLY
+LLLYSCK+
QICK+SSQICK+
Collision Induced Dissociation1) Electric potential applied to collision cell2) Ions accelerated to high kinetic energy3) Ions collide with neutral gas molecule4) Kinetic energy is converted to internal energy5) Chemical bond breakage occurs
Measure m/z values of product ions+LLLYSSQICK+
N2 collision gas
N2
N2 N2
Tandem Mass Spectrometry1st measurement (MS1) = Intact Peptide m/z2nd measurement (MS2) = Peptide fragment ion m/z
values
PRECURSOR ion 612.8 m/z(LLLYSSQICK) filtered in Q1
Q1 q2 Detector
Collision Induced Dissociation (CID or HCD)
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
matrixscience.com
peptide bonds
• Peptide Backbone has 3 Bond Types (peptide bond is the weakest of the 3 bonds)• Bond Breakage Yields Complimentary Ion Types, e.g., b- and y-type
Predictable Fragment Ions Types from Peptide Dissociation
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
Peptide MQIFVKTLTK • 604.8572 m/z precursor ion, monoisotopic• 1208.7077 Intact Peptide Mass, monoisotopic
m/z b series y series m/z1062.6 MQIFVKTLT K 147.1961.6 MQIFVKTL TK 248.1848.5 MQIFVKT LTK 361.3747.4 MQIFVK TLTK 462.3619.3 MQIFV KTLTK 590.4520.3 MQIF VKTLTK 689.5373.2 MQI FVKTLTK 836.5260.1 MQ IFVKTLTK 949.6132.0 M QIFVKTLTK 1077.7
y1y2y3y4y5y6y7y8y9
b9b8b7b6b5b4b3b2b1
Peptide Fragment Ions (or product ions)
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
Peptide Tandem MS can provide Unambiguous Evidence for Location of
Amino Acid Modifications
Rule: Experimental data must contain fragment ions that provide site localization evidence
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
MQIFVKTLTK MWmono = 1208.7077MQIFVKTpLTK MWmono = 1288.6740 (phosphorylation +80 Da = HPO3)
Mass shifts will occur only in fragments containing the phos-Thr, therefore location of MODIFICATION can be pinpointed
m/z b series y series m/z1062.6 MQIFVKTLT K 147.1961.6 MQIFVKTL TK 248.1848.5 MQIFVKT LTK 361.3747.4 MQIFVK TLTK 462.3619.3 MQIFV KTLTK 590.4520.3 MQIF VKTLTK 689.5373.2 MQI FVKTLTK 836.5260.1 MQ IFVKTLTK 949.6132.0 M QIFVKTLTK 1077.7
Tandem MS (or MS/MS) for Identification of Amino Acid Post-translational Modification (PTM) Site
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
+80+80+80+80+80+80
+80+80+80
m/z b series y series m/z1062.6 MQIFVKTLT K 147.1961.6 MQIFVKTL TK 248.1848.5 MQIFVKT LTK 361.3747.4 MQIFVK TLTK 462.3619.3 MQIFV KTLTK 590.4520.3 MQIF VKTLTK 689.5373.2 MQI FVKTLTK 836.5260.1 MQ IFVKTLTK 949.6132.0 M QIFVKTLTK 1077.7
Mass shifts will occur only in fragments containing the phos-Thr, therefore location of MODIFICATION can be pinpointed
Predictable Mass Shifts for Phosphorylated FragmentsMQIFVKTpLTK
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
m/z b series y series m/z1142.6 MQIFVKTpLT K 147.11041.5 MQIFVKTpL TK 248.1928.4 MQIFVKTp LTK 361.3747.4 MQIFVK TpLTK 542.3619.3 MQIFV KTpLTK 670.4520.3 MQIF VKTpLTK 769.4373.2 MQI FVKTpLTK 919.5260.1 MQ IFVKTpLTK 1029.6132.0 M QIFVKTpLTK 1157.6
MQIFVKTpLTK (+80 = HPO3); MWmono = 1288.67
Mass shifts will occur only in fragments containing the phos-Thr(bold), therefore location of MODIFICATION can be pinpointed
Predictable Mass Shifts for Phosphorylated Fragments
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
EXAMPLE: MS2 Spectrum and Confirmed Peptide Match a=3.56742302613418490e-004, t0=4.31796646529983260e+001
100 200 300 400 500 600 700 800 900 1000 1100 1200m/z, amu
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2116
199.1716
86.0972307.1400
136.0725
249.1534 722.3468340.2637
147.1153 420.2225 885.3852 998.4837277.1471
289.1373 635.3242
a1
a2
y8(2+)
b2
y9
b3
y8
a4b4
y7
a5
y6
y5
y4
y3
y2
a3-NH3(2+) y7(2+)
y1
L L L Y S S Q I Ccam KProtein ID: Tyrosine-protein kinase JAK3 (human)
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
Intact Protein Electrospray Mass Spectrometry
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
Intact Protein Electrospray - Mass Spectrum
200 1000 2000m/z
+71766.6
+81545.7
+91374.2
+101236.9
+111124.6
+121031.0
+13951.8
+14884.0
+15825.0
+16772.4
+17727.5
0
50
100
Rela
tive
Abun
danc
e PROTEIN CHARGE SERIES
10000mass12500 15000
0
50
100
Rela
tive
Abun
danc
e
12359 +/- 2
Mathematically deconvoluted Data Protein MW
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
Intact Protein ESI-MS Cytochrome C, 12361 Da
mz = 12361 + 8
8 = 1546
+8
mz = 12361 + 12
12 = 1031+12
1031.0
200 1000 2000m/z
1545.7
0
50
100
Rel
ativ
e Ab
unda
nce
NH3+
COOH
H+
H+H+ H+
H+H+H+
Charge = +8
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
Intact Protein Electrospray-MSInfusion of Solubilized Relatively Pure Protein
• Relatively pure sample• 20 – 50 µM protein concentration• Detergent and salt free solution• Typical solvent: 50:50, acetonitrile:water, 0.1% formic
acid• Difficult (but possible) to achieve high quality data• Non-covalent interactions are retained with ESI (not
MALDI), usually with neutral/basic buffer system and a lot of trial and error
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
1600 1800 2000 2200
0
2000
4000
6000
8000
17921730
1858
16721930
1618 2007
18151753 209018821568
m/z
Inte
nsity
, cou
nts Intermediate 1
Protein
Glycosylated Intact Protein ESI Mass Spec
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
Intermediate 1: Deconvoluted MS DataUsing Bayesian Reconstruct Tool; Theoretical mass = 50140.01 Da; error=4 ppm
5.00e4 5.05e4 5.10e4Mass, amu
50140
50180
5079750229 5049749849
Inte
nsity
, cps
1.0e5
8.0e4
6.0e4
4.0e4
2.0e4
050943
.23
∆ 656
+K
+K
Sialic acid
∆ 291
Fucose
∆146
NAcNeu|
Gal|
GlcNAc