The Perfinity Workstation
What about function and activity?
Isoforms
PTMs
CD44
Non-small cell lung cancer
A single insertion differentiates
metastatic from benign cancer
Kinase (enzyme) stuck in “on”
position
Unregulated growth
TFSTVHPIPDE
Prostate Specific Antigen (PSA)
61-78% false positive rate by immunoassay
At least 4 isoforms
Mikolajczyk et al. (2004), Clin. Biochem. 37:519-528.
E
su
rfa
Antibodies have trouble differentiating
between little differences in proteins
ce
How serious is the isoform problem?
There are ~30,000 genes in the human genome.
Humans express >300,000 protein species.
Proteins can exist as 10-50 isoforms simultaneously, many differing in
biological activity.
Single amino acid variants can cause a disease.
Federal regulatory agencies are concerned about
which isoforms are biologically active and
whether biological averages of all isoforms are being reported.
Differentiating between isoforms is an
essential part of quantification and
establishing structure-function relationships.
Why Is There Not Broad Application?
Protein sample preparation bottleneck
Current approaches are
a) Complex
b) Multifaceted
c) Often irreproducible
Automating Sample Preparation
Delivers…
1) Quality – Exceptional reproducibility!!!
2) Speed – 4 minute digests
3) Value – Costs ½ as much
Automation of Established
Technologies/Best Practices
ClK
K
R
Na+
Na+
R
Cl-
ClNa
+
The Workstation Platform
1. Two, independent centrally controlled pumping systems.
1 1
0
1 1
0
6
6
1 1
0
Quat
6
PDA
PDA
UFLC
Pump A
Pump B
UFLC
FRC
CBM
UFLC
2. The pumping systems are capable of flow rates ranging from 10 uL/min to 10
mL/min and an operating pressure of 10,000 psi.
3. Column I.D. can range from 300 um to 4.6 mm.
4. Detection is occurring at multiple points in the analysis.
5. Auto-sampler holds reagents and samples
6. Sample analysis is totally automated.
7. Multiple patented technologies integrated into a single platform.
Digestion of Insulin Under Standard Conditions
uV
5000000
4500000
4000000
GFFYPTK
Undigested Protein
3500000
30 minutes
3000000
1 hour
3 hours
2500000
2000000
6 hours
1500000
12 hours
1000000
500000
18 hours
24 hours
0
4.0
4.5
5.0
Protein
Product
Sample:
Column :
Mobile Phase A:
Mobile Phase B:
Detection:
5.5
6.0
5ug insulin
HALO 2.1x100mm RPC
2% ACN, 98% water, 0.1%
Formic Acid
90% ACN, 10% water, 0.1%
Formic Acid
UV/VIS at 214nm
6.5
7.0
min
Introduces >10% Variability
Slept in
Couldn’t sleep
9
With Perfinity Complete Digest in 8 Minutes or Less
uV
2750000 Data1:Digest Insulin 50uL 4-185-03 400uLmin 60C May 17 Run 1.lcd Detector A:214nm
Data2:Digest Insulin 50uL 4-185-03 200uLmin 60C May 17 Run 3.lcd Detector A:214nm
Data3:Digest Insulin 50uL 4-185-03 100uLmin 60C May 17 Run 2.lcd Detector A:214nm
2500000 Data5:Digest Insulin 50uL 4-185-03 50uLmin 60C May 17 Run 3.lcd Detector A:214nm
2250000
Undigested
Protein
2000000
1750000
GFFYTPK
1500000
1250000
1 minute
1000000
2 minutes
4 minutes
8 minutes
750000
500000
250000
0
19.0
20.0
21.0
22.0
23.0
24.0
25.0
26.0
min
Protein Product
Sample:
Column 1:
Column 2:
Mobile Phase A:
Mobile Phase B:
Detection:
5ug insulin
Perfinity Optimized Trypsin Column
HALO 2.1x100mm RPC
2% ACN, 98% water, 0.1% Formic Acid
90% ACN, 10% water, 0.1% Formic Acid
UV/VIS at 214nm
What about something
a little more complex?
uV
Data1:10uL R&A Tr 4-139-03 2pt1mm Halo 1 x20 300A GRD R8 Apr 1.lcd PDA Ch1:214nm,4nm(1.00)
Data2:10uL R&A Tr 4-139-03 2pt1mm Halo 1 x20 300A GRD R7 Apr 1.lcd PDA Ch1:214nm,4nm(1.00)
Data3:10uL R&A Tr 4-139-03 2pt1mm Halo 1 x20 300A GRD R6 Apr 1.lcd PDA Ch1:214nm,4nm(1.00)
-200000 Data4:10uL R&A Tr 4-139-03 2pt1mm Halo 1 x20 300A GRD R5 Apr 1.lcd PDA Ch1:214nm,4nm(1.00)
Data5:10uL R&A Tr 4-139-03 2pt1mm Halo 1 x20 300A GRD R4 Apr 1.lcd PDA Ch1:214nm,4nm(1.00)
Data6:10uL R&A Tr 4-139-03 2pt1mm Halo 1 x20 300A GRD R3 Apr 1.lcd PDA Ch1:214nm,4nm(1.00)
Time
-300000
Run 1
Run 2
Run 3
Average
StDev
CV(%)
20.0
77551
72199
80057
76602.33
4014
5.24
20.4
123633
106875
121037
117181.7
9020
7.70
20.9
101131
108713
105673
105172.3
3816
3.63
21.1
164803
168049
167296
166716
1699
1.02
21.5
261128
236951
234215
244098
14812
6.07
22.2
290171
290735
288299
289735
1275
0.44
22.4
186600
178884
178861
181448.3
4461
2.46
22.7
143964
144298
142517
143593
947
0.66
22.9
104212
114875
110346
109811
5352
4.87
23.2
267686
255772
249757
257738.3
9125
3.54
23.4
130573
124909
129993
128491.7
3116
2.43
23.5
53220
48288
49150
50219.33
2634
5.25
23.6
31610
32296
31467
31791
443
1.39
23.8
102430
105401
101824
103218.3
1914
1.85
24.0
49022
56231
53091
52781.33
3614
6.85
24.2
91279
86549
78148
85325.33
6650
7.79
24.4
85161
85883
82149
84397.67
1981
2.35
24.8
241992
243983
250666
245547
4544
1.85
25.0
483586
471184
467367
474045.7
8480
1.79
25.5
34326
36274
31764
34121.33
2262
6.63
-400000
-500000
-600000
-700000
-800000
-900000
-1000000
20.0
22.5
25.0
27.5
30.0
32.5
35.0
37.5
40.0
•Reduced and alkylated transferrin
•CV’s of first 20 peptides of first 3 runs
Average CV = 3.69%
42% Sequence Coverage
Sample process time = 44 minutes
42.5
min
Average CV =
3.69
How does it compare?
Time
uV
-880000 Data1:Solution Dig Transferrin 100uL 4-158-04.lcd PDA Ch1:214nm,4nm(1.00)
Data2:Solution Dig Transferrin 100uL 4-158-06.lcd PDA Ch1:214nm,4nm(1.00)
Data3:Solution Dig Transferrin 100uL 4-158-05.lcd PDA Ch1:214nm,4nm(1.00)
-890000
-900000
-910000
-920000
Run 1
Run 2
Run 3
Average
StDev
CV(%)
16.55
852387
1149317
1161884
1054529
175173
16.61
17.00
3734784
4317305
4307224
4119771
333447
8.09
21.96
271377
298492
328634
299501
28642
9.56
22.99
428229
505473
520549
484750
49526
10.22
24.02
277636
311323
337541
308833
30030
9.72
24.35
451355
498854
561522
503910
55257
10.97
24.84
273835
316991
319944
303590
25811
8.50
25.73
304591
278655
314850
299365
18655
6.23
27.75
282650
346649
387876
339058
53022
15.64
29.39
276798
258535
270154
268496
9244
3.44
32.59
477172
557145
597765
544027
61357
11.28
33.55
288461
455468
509485
417805
115225
27.58
34.19
344019
271344
269006
294790
42650
14.47
34.58
510189
256581
319090
361953
132126
36.50
35.61
250712
291287
288096
276698
22561
8.15
36.17
386645
451144
489667
442485
52054
11.76
36.88
250506
280509
339414
290143
45230
15.59
37.41
762001
872341
920352
851565
81194
9.53
38.02
543185
680836
698035
640685
84875
13.25
39.35
580299
697772
742210
673427
83656
12.42
-930000
-940000
-950000
-960000
-970000
-980000
-990000
-1000000
-1010000
-1020000
15.0
20.0
25.0
30.0
35.0
40.0
45.0
min
•Reduced and alkylated transferrin
•CV’s of first 25 peptides of first 3 runs
Average CV = 12.98%
20% Sequence Coverage
Sample process time = 25 hours
Average CV =
12.98
What about carry-over?
uV
-100000 Data1:10uL R&A Tr 4-139-03 1mm Halo 2pt1 x20 300A GRD no urea R1 Apr 1.lcd PDA Ch1:214nm,4nm(1.00)
Data2:blank digestion run.lcd PDA Ch1:214nm,4nm(1.00)
-125000
-150000
-175000
-200000
-225000
-250000
-275000
-300000
-325000
-350000
15.0
20.0
25.0
30.0
35.0
40.0
min
How Did We Get Rid of Carry-over?
By Maximizing Recovery!!!
uV
Data1:RAQ Hb S 25ug 4D 2-50 in 20 40C.lcd Detector A:214nm
500000 Data2:RAQ Hb S 25ug 4D 2-50 in 20 40C 50mM Tris.lcd Detector A:214nm
450000
400000
Perfinity Digest Buffer
350000
300000
250000
200000
150000
100000
50000
0
Standard Tris buffer
-50000
-100000
-150000
10.0
12.5
15.0
17.5
20.0
22.5
25.0 min
Confirmed
uV
Data1:RAQ
Data2:RAQ
650000 Data3:RAQ
Data4:RAQ
600000
Hb
Hb
Hb
Hb
S
S
S
S
25ug
25ug
25ug
25ug
4D
4D
4D
4D
2-50
2-50
2-50
2-50
in
in
in
in
20
20
20
20
40C.lcd Detector A:214nm
40C 50mM Tris.lcd Detector A:214nm
40C R 2.lcd Detector A:214nm
40C 50mM Tris R2.lcd Detector A:214nm
550000
500000
450000
400000
350000
300000
250000
200000
150000
100000
50000
0
7.5
10.0
12.5
15.0
17.5
20.0
22.5
min
You are Taking
An Uncompromising Approach
High selectivity (+)
•Antibodies can be used to isolate
proteins from biological extracts.
G
G
N
N
M
M
R
L
R
L
H
H
H
H
D
D
M
A
N
Q
M
A
N
Q
K
K
L
L
Poor Resolution (-)
•Immunological contact areas
(epitopes) are very small
Chromatography
Poor selectivity (-)
•Proteins must be extracted or samples
fractionated prior to analysis
Reverse phase separation of insulin variants,
some of which differ by a single amino acid.
High resolution (+)
•Similar proteins differing by small
changes in structure can easily be
resolved.
High volume washing to minimize non-specific binding
1.6
120
Affinity sorbent stationary phase = -Protein G:Ab:Ag; 2 column volume wash.
Antigen released from Ab with pH 2.5 eluent
1.4
100
1.2
Transferrin
0.8
60
Without abundant
protein removal
0.6
80
Low affinity proteins
are still bound to the
protein G column.
Immunoglobulins
40
0.4
20
0.2
0.0
0
0
2
4
6
8
10
12
14
16
Minutes
18
20
22
24
26
28
30
%
AU
1.0
The value of high speed, high volume washing
Affinity sorbent stationary phase = -Protein G:Ab:Ag; 40 column volume wash
Antigen released from Ab with pH 2.5 eluent
Transferrin
The binding constant of
transferrin is so much larger than
the non-specifically bound
proteins they are washed away at
high wash volumes.
Desorption of
weakly bound
proteins can occur
with 60 to 120
seconds of washing.
HSA
A More Reliable Immunoassay
A
sample
soln.
[B-Ab:Ag] + -[Av4]
Kd=
10-15M
avidin
H+
column
-[Av4:B-Ab:Ag]
= 1 fm
avidin
column
-[Av4:B-Ab] + [Ag]
RPC
RPC
trypsin digestion
Universal
affinity column
Enhancing Capture Kinetics
Antigen(s) eluting from avidin column.
•Allow binding to occur in solution
•Force antigens through an -Av tunnel
transferrin
RPC
B-Ab:Ag B-Ab:Ag B-Ab:Ag
B
-Av
Av-Av
Av-Av
Av- 100 nm -
[Av] 100 – 1000 times larger than [B-Ab]
minute
Amount (ng)
5000
2500
1250
500
75
10
Standard Curve of Hu Transferrin
Run 1 Run 2 Run 3 Average Stdev CV
210.1 225.8 222.8
219.6
8.3
3.8
202.6 211.0 218.8
210.8
8.1
3.8
175.9 178.1 179.5
177.8
1.8
1.0
131.6 130.5 130.9
131.0
0.6
0.4
62.7 62.6 64.4
63.2
1.0
1.6
28.7 30.2 27.9
28.9
1.2
4.0
Quantification using the Perfinity Workstation
Sample
Ag(1-10)
R-Ab(1-10)
RPC
SILAC - Two samples are added
to a single reaction vessel. Ab
selection done at protein level
P2
Label free/SRM. No change in
standard work flow is needed.
P3
P1
UV/Vis
immunosorbent
RPC
B
MALDIMS
auto-sampler
A
UV/Vis
ESI-MS
waste
oven
nd
2
dimension LC
refrigerated
waste
samples
st
1 dimension LC
MRM – Ab based selection can be
done at either the protein or peptide
level. Labeled peptides are added
just before RPC.
GIST/iTRAQ. Does not fit this
work flow very well. Proteolysis
and isotope coding must be done
before Ab selection. This means
all Ab selection is done at the
peptide (or hapten) level.
Quantitation
Replicate
Cal 10
Cal 100
Cal 1000
Cal 10000
1
486
1983
18688
209009
2
724
1970
19195
210932
3
637
1857
18595
204153
Mean:
615.6667
1936.667
18826
208031.3
StDev:
120.4256
69.29887
322.9288
3493.649
%CV:
19.60%
3.60%
1.70%
1.70%
Assays with CV’s <10%
uV
Data1:R&A
Data2:R&A
Data3:R&A
1000000 Data4:R&A
1010000
Hb
Hb
Hb
Hb
A
A
A
A
4-137-05
4-137-05
4-137-05
4-137-05
3
3
3
3
18
18
18
18
11
11
11
11
R4.lcd
R3.lcd
R2.lcd
R1.lcd
PDA
PDA
PDA
PDA
Ch1:214nm,4nm(1.00)
Ch1:214nm,4nm(1.00)
Ch1:214nm,4nm(1.00)
Ch1:214nm,4nm(1.00)
990000
980000
970000
960000
950000
940000
930000
920000
910000
900000
890000
880000
870000
860000
17.5
Run 1
Run 2
Run 3
Run 4
Average
StDev
CV(%)
1
814
793
745
865
804
50
6.2
2
3
4
1355
942 1305
1283
933 1261
1269
874 1218
1428 1022 1284
1334
943 1267
73
61
37
5.5
6.5
2.9
20.0
5
1610
1336
1414
1601
1490
137
9.2
6
2469
2287
2310
2362
2357
81
3.4
22.5
Peak Number
7
8
9
2145 2353 1233
1803 2052 1131
1886 2084 1116
2064 2292 1223
1975 2195 1176
157
150
61
8.0
6.8
5.2
25.0
10
1535
1376
1485
1598
1499
94
6.3
11
1962
2005
1941
2245
2038
140
6.9
min
12
2805
2516
2648
2889
2715
166
6.1
13
714
649
664
789
704
63
9.0
14
439
378
393
454
416
36
8.7
15
1215
1012
1035
1156
1105
97
8.8
More of the Same
Run 5
Run 10
Run 15
Run 20
Personalize Your Method
Create your Batch
93% Sequence Coverage of Transferrin
RT: 0.00 - 20.00
4.57
100
95
90
85
80
75
70
65
11.65
60
MRLAVGALLV
FRDHMKSVIP
LDAGLVYDAY
KKDSGFQMNQ
PRKPLEKAVA
LNQYFGYSGA
QYELLCLDNT
LIWELLNQAQ
FLKVPPRMDA
VKWCALSHHE
MNGEADAMSL
PEAGYFAIAV
IPMGLLYNKI
RCLVEKGDVA
LCLDGTRKPV
RQQQHLFGSN
HDRNTYEKYL
CAVLGLCLAV
SDGPSVACVK
LAPNNLKPVV
LRGKKSCHTG
NFFSGSCAPC
FKCLKDGAGD
RKPVDEYKDC
EHFGKDKSKE
KMYLGYEYVT
RLKCDEWSVN
DGGFVYIAGK
VKKSASDLTW
NHCRFDEFFS
FVKHQTVPQN
EEYANCHLAR
VTDCSGNFCL
GEEYVKAVGN
PDKTVRWCAV SEHEATKCQS
KASYLDCIRA IAANEADAVT
AEFYGSKEDP QTFYYAVAVV
LGRSAGWNIP IGLLYCDLPE
ADGTDFPQLC QLCPGCGCST
VAFVKHSTIF ENLANKADRD
HLAQVPSHTV VARSMGGKED
NL: 1.42E8
FQLFSSPHGK
DLLFKDSAHG
Base Peak m/z=
300.00-2000.00 MS
AIRNLREGTC
PEAPTDECKP
TransferrinOnlineDigest10uLof1
SVGKIECVSA
ETTEDCIAKI
00ngmL-19Oct2011-1
CGLVPVLAEN YNKSDNCEDT
DNLKGKKSCH TAVGRTAGWN
GLNLCEPNNK EGYYGYTGAF
TGGKNPDPWA KNLNEKDYEL
APNHAVVTRK DKEACVHKIL
FRSETKDLLF RDDTVCLAKL
LRKCSTSSLL EACTFRRP
55
9.64
50
4.65
9.45
45
17.55
40
9.37
35
8.89
30
25
20
17.71
15
11.31
10
6.53
3.85
7.51
12.56
2.99
5
0.09
15.39
13.04
4.87
2.46
14.09
19.61
16.58
0
0
2
4
6
8
10
Time (min)
12
14
16
18
20
SRM of a specific peptide -MYLGYEYVTAIR
RT: 0.00 - 20.00
RT: 0.00 - 20.00
10.86
100
4.57
100
NL: 1.42E8
Base Peak m/z=
300.00-2000.00 MS
TransferrinOnlineDigest10uLof1
00ngmL-19Oct2011-1
95
95
90
90
85
NL: 1.88E6
m/z= 739.50-740.50 F: ITMS
+ c ESI Full ms
[300.00-2000.00] MS
TransferrinOnlineDigest10uLof100ngmL19Oct2011-1
MS2 Spectrum for 1
Peptide
85
80
80
75
70
75
65
70
60
55
11.65
65
9.64
50
4.65
60
45
9.45
17.55
40
55
9.37
35
8.89
50
30
25
45
20
17.71
40
15
11.31
10
6.53
3.85
2.99
35
5
0.09
0
2
12.56
30
4
14.09
15.39
13.04
4.87
2.46
0
7.51
6
8
10
Time (min)
12
19.61
16.58
14
16
18
20
25
20
15
9.66
14.73
10
6.27
5
2.84
4.57 5.45
3.02
9.48
9.94
8.95
7.11
11.57
11.70
14.28
12
14
15.62 16.82 18.22
0
0
2
4
6
8
10
Time (min)
16
18
20
Quantitation
Standard Curve of Insulin
C-terminal Peptide
y = 20.843x - 537.99
R² = 0.9999
250000
200000
150000
100000
50000
0
0
2000
4000
6000
8000
10000
12000
Insulin Concentration (ng/mL)
Replicate
10
100
1000
10000
Run 1
486
1983
18688
209009
Run 2
724
1970
19195
210932
Run 3
637
1857
18595
204153
Mean:
616
1937
18826
208031
StDev:
120
69
323
3494
%CV:
19.56
3.60%
1.70%
1.70%
Isoform Identification
MASCOT Results
Rapid identification of VHLTPVEK
sickle cell peptide isoform by IT-TOF
•91% sequence coverage
•Rapid identification of sickle cell variants
•Fully automated extraction, digestion and analysis
Selective Ion Monitoring
A wide variety of oxidative
stress induced posttranslational modifications
(OSi~PTMs) can occur in
the same protein at the
same time.
Carbonylation sites are revealed by MS/MS
Y1 ion corresponds to the mass of biotinylated oxidized arginine (mass =374.19)
Arginine oxidation product
MS/MS Fragmentation of GLEWLGR*, a peptide from the immunoglobulin heavy
chain variable region showing the oxidation of C-terminal arginine
Distribution of the oxidized proteins in
tissues of 30-35 year old males
Number of proteins
Most protein are in plasma as a result of cell
death. In essence we are performing an
autopsy on a cell that just died.
Madian, A. G.; Regnier, F. E., Profiling Carbonylated Proteins in Human Plasma. J. Proteome Res. 2010, 9, (3), 1330-1343.