Characterization of Intact
Proteins by ESI-LC/MS
1
Characterization of Intact Proteins
• Recombinant Proteins
– integrity of construct
– extent
t t and
d ID off post-translational
tt
l ti
l or other
th
modifications
• N
Native
ti P
Proteins
t i
• Protein-Protein or Protein-Small
Molecule Interactions
p
sequencing
q
g
• “Top-down”
2
Challenges
g
• Concentration
– usua
usually
y need
eed p
pmols
oso
of relatively
e a e y pu
pure
e
protein
• Sample Handling
– buffer components
• Problematic classes of proteins
– membrane proteins; very large proteins;
highly folded
folded, disulfide rich proteins
• Other problems
– aggregation;
ti
precipitation
i it ti
3
Typical Biological Buffer
Components
• Buffers: HEPES, Tris, TBS, PBS
• Detergents: SDS, Triton, NP-40, CHAPS (see
Appendix)
• Chaotropes: guanidine,
guanidine urea
• Reductants: DTT, 2-mercaptoethanol, ascorbic
acid
• Salts: NaCl, KCl
• Chelators: EDTA
• Protease Inhibitors: aprotinin, leupeptin, PMSF,
pepstatin
• Stabilizers: glycerol, mannitol, PEG
• Bacteriostats: NaN3
4
Electrospray vs MALDI
• Electrospray
– direct coupling to LC for on-line
desalting/preconcentration
– multiple charging allows analysis of proteins with MW’s
greater
g
eate than
t a the
t e mass
ass range
a ge o
of tthe
e mass
ass spect
spectrometer
o ete
– resolution of species with relatively small molecular
weight differences (methylation, oxidation, etc.)
– soft
ft ionization
i i ti (preserve
(
some noncovalent
l t
interactions)
• MALDI
– off-line sample prep
– relatively low resolution protein spectra
– more tolerant of salts than ESI
• ESI/MALDI often complementary
5
Useful Conversions
•
•
•
•
1 mM = 1000 µM
1 µM = 1 pmol/µL
1 mg/mL = 1 µg/µL
T convertt mg/mL
To
/ L or µg/µL
/ L tto pmol/µL:
l/ L
pmol/µL = (mg/mL x 106)/protein MW
Example: 1 mg/mL of a 30 kDa protein
[(1 mg/mL) x 106)/30,000] = 33.3 µM
6
Electrospray Produces Multiply
Charged Ions
• In + ion mode, 10’s to 100’s of charges via
protonation or adduct formation (Na+, K+)
• Parameters for optimization include:
–
–
–
–
cone/skimmer voltages (charge stripping)
heated capillary temperature
desolvation gas flow
b ff composition
buffer
iti (f
(for noncovalent
l t interactions)
i t
ti
)
• Usually use volatile buffers (NH4HCO3) in the pH 7-7.5
range to preserve interactions
7
ESI of Myoglobin
y g
((MW ~ 17 kDa))
myo cal
q3t00270 370 (7.032) Sm (Mn, 4x8.00); Cm (363:391)
942.8212 998.2308
100
myo cal
q3t00270 370 (7.032) Sm (Mn, 4x8.00); Cm (363:391)
1060.5437
100
TOF MS ES+
518
TOF MS ES+
585
1060.54
893.2551
1060.5437
%
848.6454
1131.1853
1063.86
1067.16
1063.8629
1067.1605
1211.9092
0
m/z
1056
1057
1058
1059
1060
1061
1062
1063
1064
1065
1066
1067
1068
1069
1070
1071
1072
1073
1074
1075
1884.5973
%
1305.0601
808.2803
1696.2451
2120.0256
1413.7062
1542.1244
1890.4971
771.6001
2126.6787
1309.1267
1701.5554
1418.1078
1546.9144
2422.7205
2430.3567
2133.3044
1896.3701
1422.5162
0
m/z
700
800
900
1000
1100
1200
1300
1400
1500
1600
1700
1800
1900
2000
2100
2200
2300
2400
8
Calculating Protein MW’s from
Adj
Adjacent
t Charge
Ch
St
States
t
M = molecular weight
g of p
protein
z = charge state
In
ntensity
Y
X = (M+z)/z
Y = (M+z+1)/(z+1)
X
m/z
To calculate the charge state (z) of X,
zx = (Y-1)/(X-Y)
(Y 1)/(X Y)
To calculate the molecular weight of the protein,
M = (X * zx) – zx = (Y * zy) - zy
9
Calculating Protein MW’s:
M
Myoglobin
l bi E
Example
l
X = 998.23
Y = 942.82
zx = (Y-1)/(X-Y)
= (942.82
(942 82 - 1)/(998.23
1)/(998 23 - 942.82)
942 82) = 16
16.997
997
∴ X = 17 and Y = 18
∴,
M = ((X * zx) – zx = 998.23 * 17 - 17 = 16952.91
Predicted MW of Myoglobin = 16951.5
If calculated for multiple adjacent charge states, an average
MW and standard deviation may be determined.
10
***Must be sure X and Y are from same series!!!!!
Calculating Protein MW’s:
A t
Automated
t d Calculation
C l l ti
myo cal
q3t00270 370 (7.032) AM (Cen,8, 80.00, Ht,5000.0,0.00,1.00); Sm (Mn, 4x8.00); Cm (363:391)
A18
A17
100
942.8455
TOF MS ES+
584
A:
B:
998.2512
A19
893.2781
16952.96±0.20
17005.74±0.19
A16
1060.5780
A15
1131.2175
A20
848.6612
A14
1211.9413
%
A9
1884.6340
A13
1305.0819
A21
808.2936
A10
1696.2798
A12
1413.7439
A8
2120.0789
A11
1542.1663
B9
1890.5209
B13
1309.1353
B12
1418.1470
B8
2126.7100
B10
1701.5680
B11
1546.9542
1313.2405 1422.5557
1551.8065
1321.4646
1431 3927
1431.3927
1694 6608
1694.6608
2133.3728
1896.4167
1706.9254
1718.8639
1902 3551
1902.3551
2117 8416
2117.8416
2140.1606
0
m/z
800
900
1000
1100
1200
1300
1400
1500
1600
1700
1800
1900
2000
2100
2200
2300
11
Calculating Protein MW’s:
D
Deconvoluting
l ti ffrom m/z
/ tto MW
myo cal
myo cal
q3t00270 370 (7.032) Sm (Mn, 2x3.00); Cm (361:396)
942.82 998.22
100
TOF MS ES+
675
q3t00270 370 (7.032) M1 [Ev-151748,It12] (Gs,0.750,724:2357,1.00,L30,R10); Sm (Mn, 2x3
16953.00
4.88e4
100
893.27
1060.53
MaxEnt1
M
E t1
Deconvolution
1131.20
Measured:
M
d 16953
Predicted: 16951.5
Error = 1.5 Da =
0 009%
0.009%
1211.94
1884 47
1884.47
%
%
1305.06
1696.25
2120.03
1413.74
1542.09
1890.41
2126.77
17006.00
1701.49
1896.32
2133.23
16934.00 17058.00
0
m/z
800
1000
1200
1400
1600
1800
2000
2200
0
15000
17951.00
mass
16000
17000
18000
19000
12
Deconvoluting from m/z to MW:
Artifacts from Deconvolution
myo cal
q3t00270 370 (7.032) M1 [Ev-222603,It5] (Gs,0.750,716:2461,2.00,L30,R10); Sm (Mn, 2x3.00); Cm (360:391)
16952.00
100
TOF MS ES+
6.77e3
Myoglobin
Artifact peaks at 1/2X, 2X, 3X, etc.
Look back at raw data to rule out artifacts.
%
8476.00
17006.00
33906.00
17058.00
8502.00
11302.00
9418 00
9418.00
0
8000
13186.00
15070.00
50 0 00
25430.00
19072.00
21192.00
22604.00
23734.00
26370.00
34012.00
28254.00 29668.00
mass
10000
12000
14000
16000
18000
20000
22000
24000
26000
28000
30000
32000
34000
13
Deconvoluting from m/z to MW:
Artifacts can be identified by examining raw data
myo cal
q3t00270 370 (7.032) AM (Cen,8, 80.00, Ht,5000.0,0.00,1.00); Sm (Mn, 4x8.00); Cm (363:391)
A18
A17
100
942.8455
998.2512
A19
893.2781
TOF MS ES+
584
A:
B:
Arrows show where ions should be for
a protein at MW 33906
A16
1060.5780
16952.96±0.20
17005.74±0.19
A15
1131.2175
A20
848.6612
A14
1211.9413
%
A9
1884.6340
A13
1305.0819
A21
808.2936
A10
1696.2798
A12
1413.7439
A8
2120.0789
A11
1542.1663
B9
1890.5209
B13
1309.1353
B12
1418.1470
B8
2126.7100
B10
1701.5680
B11
1546.9542
1313.2405 1422.5557
1551.8065
1321.4646
1431 3927
1431.3927
1694 6608
1694.6608
2133.3728
1896.4167
1706.9254
1718.8639
1902 3551
1902.3551
2117 8416
2117.8416
2140.1606
0
m/z
800
900
1000
1100
1200
1300
1400
1500
1600
1700
1800
1900
2000
2100
2200
2300
14
Deconvoluting from m/z to MW: Choosing
the m/z range
CDK 4 lot 18477-10 Holmes. B
CDK 4 lot 18477-10 Holmes. B
q3t00283 267 (8.696) Sm (Mn, 3x5.00); Cm (260:276)
814.39
100
TOF MS ES+
q3t00283 267 (8.696) M1 [Ev40178,It20] (Gs,0.750,300:2500,2.00,L30,R5); Sm (Mn, 3x5.00); C
179
47.2
100
892.91
893.41
1164.53
1010.03
814.05
1221.07
776.69
MaxEnt1
1246 91
1246.91
776.38
1385.34
%
%
15
0
500
750
1000
1250
1500
1750
2000
2250
m/z 0
72500
mass
72750
73000
73250
73500
73750
74000
74250
Deconvoluting from m/z to MW: Choosing
g
the m/z range
CDK 4 lot 18477-10 Holmes. B
CDK 4 lot 18477-10 Holmes. B
TOF MS ES+q3t00283 267 (8.696) M1 [Ev-17412,It20] (Gs,0.750,1642:2490,2.00,L40,R10); Sm (Mn, 3x5.00
73266.00
982
23.9100
q3t00283 267 (8.696) Sm (Mn, 3x5.00); Cm (260:276)
1666.16
100
diphos
monophos
p
73186.00
1704.82
1745.47
FL nonphos
1785.93
1832.79
73300.00
1879.59
73106 00
73106.00
%
%
73346.00
73374.00
1929.10 1981.21
73432.00
72500.00
2036.17 2092.06
72920.00
2094.28
2153.71
73500.00
74170.00
73828.00
72772.00
2290.36
2361.88 2443.21
74006.00
73672.00
72884.00
2221.12
74384.00
72528.00
2445.01
0
1700
1800
1900
2000
2100
2200
2300
2400
m/z 0
72500
16
72750
73000
73250
73500
73750
74000
74250
mass
•
Sample Handling/Cleanup for
ESI
Sample Handling
– store samples at -20 to -80 oC
– keep on ice when in use
use, use refrigerated autosampler
– avoid multiple freeze/thaw cycles (aliquot)
• Dilution
– if protein concentration sufficiently high (mg/mL quantities)
– dilute with 50/50 MeOH/water, 1-5% formic or acetic acid
– Infuse at 1-5 µL/min
• Reversed-phase
– C2, C4, C8, C18, Poros and other polymeric resins
– perfusive resins (Poros), larger particle sizes, or short
columns can allow for higher flow rates for rapid desalting
– small particle size, silica based supports offer optimal
chromatographic resolution
– extremely hydrophobic proteins may be difficult to elute from17
more hydrophobic supports
Sample Cleanup for ESI
• Reversed-phase formats
– pipette tip desalting
• homemade, ZipTips (Millipore), step elution
– Cartridges
• Dionex, Waters, Michrom
• can gradient elute with an LC or step elute/collect for infusion
– For both tips and cartridges off-line,
•
•
•
•
wet packing with organic prior to use
equilibrate with 0.1% TFA or 0.1% formic acid
load sample slowly; dilute sample if organic is > 10% in sample
wash extensively with 0.1% formic acid; may wash with 10%
MeOH or MeCN to assist in removal of hydrophobic buffer
components
• elute
el te with
ith 80% MeOH or MeCN containing 0
0.1-1%
1 1% formic acid
18
Homemade Poros RP Perfusion Column
Upchurch ZDV
PEEK Union (P742)
Stainless Frit
Upchurch (C407)
From LC
PEEK Sleeve
Fused Silica
Blue 250µm id
PEEK Tubing (15-20 cm)
Poros 10R1
10R1, 10R2
10R2, or 10R3
Load at 50-100 µL/min
Elute at 20
20-30
30 µL/min
To MS
19
Sample Cleanup for ESI
• Ultrafiltration
– molecular weight
g cut off ((MWCO)) spin
p cartridges
g ((Amicon,,
Centricon, etc.)
– low MW species pass through membrane; species of MW >
membrane cutoff do not p
pass through
g
– MWCO ranges: 3, 10, 30, 50, and 100 kDa
– choose a MWCO ~ 1/2 the MW of the protein to be retained
– can be used for salt and detergent removal
• detergents will not pass through the membrane if concentration
> CMC
– for small protein amounts,
amounts use with caution
• irreversible adsorption to the membrane
• Size Exclusion
– spin columns (BioRad, etc.)
– higher MW components elute first, low MW components
retained
20
Sample Cleanup for ESI
• Precipitation
– for removal of detergents and other components not
compatible with mass spec or protein digestion
– Methods (See appendix for protocols)
• chloroform/methanol precipitation
• acetone
• ethanol
– Resolubilization may be a problem. Try the following:
– 50% MeOH,
MeOH 5% formic acid
– may initially add a few mL of 70% formic acid, diluting quickly to <
10% (avoid formylation of protein)
– 6M Guanidine or 4M Urea
– may use a few mL of hexafluoroisopropanol or hexafluoroacetone
(highly toxic, use only in hood, vent MS source)
• Requires a lot of protein (10s to 100s of µg’s)
21
Sample Cleanup for ESI
• Ion Exchange
– Anion Exchange
• removal of SDS
• homemade pipet tips/columns, commercial cartridges
• acidify sample to ~pH 2-3; pass sample slow over anion
exchanger
• protein passes through, negatively charged SDS binds
– Cation Exchange
• select cation exchange compatible pH
pH, elution conditions; prep
sample for compatibility with CX
• Hydrophilic Interaction
– bi
bind
d iin hi
high
h organic;
i elute
l t iin hi
high
h aqueous
– can be used for detergent removal
– may be coupled on-line with MS for analysis of membrane or
other hydrophobic proteins
22
Sample Cleanup for ESI
• Microdialysis
– Pierce Slide-A-Lyzer Mini; Amika Microdialyzer
• Affinity
Affi it Capture
C t
– Ni2+ chelate for His-Tag; Fe3+/Ga3+ chelate for
phosphoproteins; monomeric avidin for biotin-tagged
proteins; antibody binding
• Prep for noncovalent interaction studies
– Must use a buffer which preserves native conformation/
interactions
– Typically, 10-50mM ammonium acetate, pH 7.0-7.5
– May have to buffer exchange prior to MS
23
Production of
Recombinant Proteins
Point mutations, fusion protein sequences,
linker sequences,
Buffers
ff andd other
h additives,
ddi i
degradation, aggregation
Post-translational modification 24
Commonly
y Used Expression
p
Systems
y
25
Common Recombinant Protein
P ifi ti Strategies
Purification
St t i
1. GST Fusion Proteins
GST Sequence (~ 26 kD)
Protein of Interest Sequence
-COOH
Linker Sequence
(M contain
(May
t i a protease
t
site,
it
i.e., thrombin, TEV protease)
-Purify
f using an immobilized glutathione column
2. His-tagged Proteins
His Tag
MSYYHHHHHHXXXX
Protein of Interest Sequence
-COOH
-Purify using an immobilized Ni2+ column. Tag may be N-terminal or
C-terminal
26
Common Observations in Protein MS
•
•
•
•
•
•
•
Loss of N-terminal
N terminal Methionine (-131
( 131 Da)
N-terminal Acetylation (+42 Da)
Phosphorylation
p y
((+80 Da))
Glycosylation (heterogeneous, variable)
Degradation/Truncation (N- or C-terminal)
Glutathionylation of GST Fusions (+305)
Phosphogluconoylation (His-tag in E. coli, + 178 or
258)
• Mutation
• Combinations of the above
• Other
27
Phosphorylation (+80 Da)
CFM-S T90 Hassell, A
CFM-S T90 Hassell, A
q3t00134 443 (8.482) Sm (Mn, 3x5.00); Cm (437:451)
1048.64
100
TOF MS ES+q3t00134 443 (8.482) M1 [Ev-65849,It9] (Gs,0.750,1378:1898,2.00,L40,R20); Sm (Mn, 3x5.00);
37606.00
100100
95.2
37526.00
37688.00
37368.00
917.65
CFM-S T90 Hassell, A
q3t00134 443 (8.482) Sm (Mn, 3x5.00); Cm (437:451)
1710.37
100
1699.38
TOF MS ES+
8.65
37448.00
1714.03
37768.00
37288.00
1313 98
1313.98
1717.81
1695.76
%
1718.82
1720.64
1687.46
1723.58
1670.03 1675.50
893.53
1731.14 1738.72
1686.04
1742.53
%
%
37208.00
873.51
584.31
0
m/z
1670
780.44
752.45
1680
1690
1700
1710
1720
1730
37166.00
1740
MaxEnt1
1332.81
0 12 3 4 5 6
1390 90
1390.90
1443.40
580.36
1447.51
Multiple Phosphorylation
States
1505.32
553.39
0
m/z
500
750
1000
1250
1500
1750
2000
2250
0
37000
mass
37200
37400
37600
37800
28
Biotinylation (mass shift varies)
FtzF1 LBD + NHS-LC-biotin Consler, T
FtzF1 LBD + NHS-LC-biotin Consler, T
q3t00250 403 (7
(7.641)
641) Sm (Mn
(Mn, 3x5
3x5.00);
00); Cm (396:431)
1494.36
100
1426.50
1364.52
TOF MS ES+ q3t00250 403 (7
(7.641)
641) M1 [Ev
[Ev-53519,It20]
53519 It20] (Gs
(Gs,0.750,927:2495,2.00,L30,R10);
0 750 927:2495 2 00 L30 R10); Sm (Mn
(Mn, 3x5
3x5.00);
00);
31360.00
101 100
1.43e4
31022.00
1569.03 1651.56
+3
1349.80
31700.00
+5
MaxEnt1
1307.71
+4
1743.25
2nd Series:
modified His-Tag
1293.58
1762.13
1255.46
+2
1241.86
30682.00
1194.13
+6
1865.70
%
%
32040.00
1162.56
1982.29
1149.96
2091.74
1137.42
+3
2114.31
1082.41
+1
2241.04
2265.21 2413.30
30342.00
+2
2
31198.00
+4
+7
31540 00
31540.00
30860.00
32378.00
2439.37
32216.00
30000.00 30522.00
0
m/z
1000
1200
1400
1600
1800
2000
2200
2400
0
30000
32724.00
32786.00
mass
30500
31000
31500
32000
32500
29
Ni Adduct Formation After His-Tag
Purification
Treated 6M Gu, 100mM EDTA,
oC Column Temp
2M
DTT,
60
WR AKT2 138-457
Untreated
WR AKT2 138-457 Fin GDE
LCT00146
C
126 ((6.273)) M1 [Ev3504,It20] (G
(Gs,0.500,1301:1551,2.00,L33,R10);) Sm
S (Mn,
(
2x3
38336.00
1.83e3
100
LCT00157
C
110 ((5.980)) M1 [Ev-13181,It20] (G
(Gs,0.500,972:1237,2.00,L33,R10);) Sm
S (Mn,
(
2x3
38336.00
1.10e4
100
FL Protein
FL Protein
+ ~59
59 Da (Ni?)
%
+ Phos
%
38398.00
+ Phos
38414.00
38414.00
38360.00
38458.00
38478 00
38478.00
38452.00
38310.00
0
38200
mass
38300
38400
38500
0
38200
38436.00
mass
38300
38400
38500
30
Proteins that don’t fly!
• Reasons
– aggregation
– tightly
g y folded/extensive disulfide network
– size
– other
• Approach
– denature
• chaotrope/reductant (add 8M guanidine, 2M
DTT,, 37oC
• temperature (60oC)
31
Proteins that don’t fly!
PB Erb4999 no tr 60C
LCT00078 466 (8.547) Sm (Mn, 3x5.00); Cm (453:509)
831.54
100
877.51
727.79
727.46 831.20
941.38
728.12
%
TOF MS ES+
402
Standard Conditions
979.56 1091.16
825.54
1150.50
651.75
1181.91
1263.35
1409.07
0
LCT00069 475 (8.712) Sm (Mn, 3x5.00); Cm (456:515)
831.52
100
TOF MS ES+
302
727.78
Treated with 6M Guanidine
941.38 1035.47
727.45 831.19831.85
728 12
728.12
%
1150 41 1219.19
1150.41
890.55
1261.21
1406.60 1462.81
1523.70 1589.95
651.72
1741.23
0
LCT00152 120 (6.163) Sm (Mn, 3x5.00); Cm (112:141)
831.66
100
TOF MS ES
ES+
289
Column Temp = 60oC
831.31831.96
962.89
988.90
938.21
1076.05 1219.40
%
727.89
727.56
1306.41 1406.84
1523.98 1590.23
1741.58
1828.60
623.99
0
600
1924.80
32
m/z
800
1000
1200
1400
1600
1800
2000
2200
2400
2600
2800
Keys to the Analysis of Large
P
Proteins
i
• Utilize a TOF mass analyzer
– Mass Resolution > 10,000
– Internal mass correction
• Lock spray or dual spray
• Good chromatography
– Separate protein from buffer salts, modifiers,
and other smaller proteins
33
hFAS – Theoretical MW = 274,629 Da
x10 8 + TIC Scan FAS_h0005.d
2.5 1
1
2
1.5
1
0.5
0.5
1
1.5
2
2.5
3
3.5
4
Counts vs. Acquisition Time (min)
4.5
5
5.5
Measured MW = 274,631
lock mass
5 ug (20pmol) of total protein injected
Courtesy of Jon Williams, GSK
34
rFAS – Theortical MW = 273,852 Da
Measured MW = 273,854
lock mass
~7 ug (20pmol) of total protein injected
Courtesy of Jon Williams, GSK
35
Common Reasons for MW
Discrepancies
1. Submitter supplies sequence with
error(s)
( )
2. Post-translational modifications
3 Mutations to protein
3.
4. Wrong protein
36