Interfacing Mass Spectrometry with Separation Methods for ... · Peptide-Polymer Hybrid Materials...
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Interfacing Mass Spectrometry with Separation Methods for Synthetic
Pol mer Anal sisPolymer Analysis
Chrys Wesdemiotis
The University of Akron, Departments of Chemistry d P l S i Ak OH 44325and Polymer Science, Akron, OH 44325
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Mass spectrometry (MS): separation + characterizationbased on mass (m/z) measurement ( )
analytes
soft
gas-phase
Key stepionizationsoft
molecularions
MSgas phase
Separation based on mass (m/z) of molecular ions.
Composition + structure information based on the m/z values of molecular and fragment ions in MS and MS/MS spectra
fragmentMS/MS
fragmentation
New ionization methods (MALDI, ESI, APCI, DESI) have enabled
fragmentions
MS/MS
2
the MS analysis of a wide range of synthetic polymers and are now widely used for their molecular structure characterization.
Ch ll i b d l iChallenges in mass-based analysis
Polymerizations often create complex mixtures that arePolymerizations often create complex mixtures that are impossible to characterize by simple (1-D) MS due to discrimination effects (in ionization or detection).
Isobaric components and isomeric architectures cannot usuallyIsobaric components and isomeric architectures cannot usually be distinguished by m/z measurement alone.
With ESI, overlapping charge distributions complicate mass determination and hence composition assignmentsmass determination and, hence, composition assignments.
Such problems can be addressed by interfacing MS with a separation method either before ionization (LC-MS) or after ionization (IM-MS: ion mobility mass spectrometry, IM-MS).
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Chromatographic separationChromatographic separation
4
Online LC-MS of polymers
The most widely used HPLC method for synthetic polymers is gel permeation chromatography (GPC), which separates according to hydrodynamic volume (size).according to hydrodynamic volume (size).
Interactive, reverse-phase LC, which separates based on chemical composition, functionality, and mass, is less common in polymer analysis,and mass, is less common in polymer analysis, but ideally suitable for the separation of mixtures with constituents of different polarities, as found in amphiphilic polymers; it is also suitable for online LC-ESI-MS.
V S i ti B C K t N S l k E d X Li C W d i ti E J M S t 18 (2012) 113
online LC ESI MS.
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V. Scionti, B.C. Katzenmeyer, N. Solak Erdem, X. Li, C. Wesdemiotis, Eur. J. Mass Spectrom. 18 (2012) 113.N. Solak Erdem, N. Alawani, C. Wesdemiotis, Anal. Chim. Acta 808 (2014) 83-93.
PEO-glucam sesquistearate (nonionic surfactant)
R = (stearate) or HR (stearate) or H
navg ≈ 5; ~1.5 mol stearate per mol surfactant
Generally a mixture of:
PEO-glucam mono and multiple stearates PEO + stearates
Generally a mixture of:
Glucam core (C7H12O5) = isobaric with 4 x C2H4O (EO monomer).
6N. Solak Erdem, N. Alawani, C. Wesdemiotis, Anal. Chim. Acta 808 (2014) 83-93.
B.C. Katzenmeyer, S. Hague, C. Wesdemiotis, Anal. Chem., submitted.
PEO-glucam sesquistearate (nonionic surfactant)
1
PEO-glucam monostearate
2
RP-UPLC 6.48
7.83
PEO-glucam distearate
PEO-glucam
6 66
PEOmonostearate
PEOdistearate
PEO-glucam tristearate
PEOaggregates
0.41 2.74
6.669.66PEO
hydrophobicity
Solvent A: 2.55 mM NH4OAc in 97% H2O / 3% MeOH – Solvent B: MeOH – Flow rate 0.4 mL/min
0.00 2.75 5.50 8.25 11.00Time [min]
7
4 2
A / B : 100:0 → 60:40 (0-2 min); 60:40 → 40:60 (2-3 min); 40:60 → 0:100 (3-7 min); 100% MeOH (>7 min)
1
1LC-MS & LC-MS2 analysis of peak
754.4981
Accurate m/z: [M + 2NH4]2+ of(PEO)n-glucam monostearate
2+
LC-MS
6 48 min
✚ ✚ ✚ ✚ ✚ ✚ ✚ ✚ ✚ ✚ ✚ ✚ ✚ ✚ ✚
44 Da
( )n gwith n = 26
3+1+
6.48 min
m/z300 675 1050 1425✚ ✚ ✚ ✚ ✚
645 39LC-MS2
PEO-glucam645.39
333.
2
245.
2 787.544
1 stearic
[M + 2Li]2+
(n = 28)
44
PEO glucam monostearate
-284
1284.82
*311.3
1 stearicacid loss
1568.09
Da
8
100 600 1100 1600 m/z
2LC-MS & LC-MS2 analysis of an oligomer in peak
2 x 284 Dastearic acid CH2CH2O HLC-MS2
.420
3.2
loss
O
O
OCH3
OO
2 2
CH CH OOCH CH stearateH
nPEO-glucam
distearate
LC-MS
7.83 min
756.
614 291
245.
2 333
*311.3 44Da
O
O
O CH2CH2O
CH2CH2O
OCH2CH2 stearate
stearate
Hn n
n-284-284
[M + 2Li]2+614.291
790.
116
506.
867
222.
553
898.549 [M + 2Li](n = 29)
200 400 600 800 1000 1200 1400 1600 1800 m/z
11512
Complete mixture separation & characterization based on polarity
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Complete mixture separation & characterization based on polarity.
PEGylated Substance P
+ ++ +
substance PMethoxy & succinimide
functionalized
PEGylated substance P (peptide-polymer
conjugate)
PEG
R-P-K-P-Q-Q-F-F-G-L-M
Potential PEGylation sites
10
14.52PEGylated Substance PmPEG
15.11
RP-UPLC
Substance PPEGylated Substance P
12.00 14.00 16.00
12.50
15.11
LC-MS spectra after post-column addition
of triethyl amine
LC-MS
14.52 min
LC-MS
15.11 min
2768.24442657.6624 2834.8101
2+
R-P-K-P-Q-Q-F-F-G-L-M
mPEGmPEG
3+
4001.6040
3957.48193236.06963048.8323
2922.39453885.1628
3290.9590
4104.9531
4251.3628
2613.6057
2591.12572526.0054
2460.0168
2900.3440
2988.3848
3054.40043076.9060
3142.9180
4+
11
2790.25442745.6855
2723.6414
2834.3010 LC-MS2635.6733
2613.64332591.6313
2591.1072
2526 0239
2900.3044
2944.8286
2988.34473010.3838
3054 4204
2+
14.52 min
2526.0239
2481.5190
2460.03492438.04492393.9834
3054.4204
3076.4368
3142.4644
3186.45733208.5061
PEGylated Substance P
2300 2400 2500 2600 2700 2800 2900 3000 3100 3200 3300
LC-MS spectra after post-column addition
of triethyl amine
3957.4819
3928.2109
4104.9531
4060.84674133.9053 4236.4048
4178.6450
4251 3628
3+
LC-MS
15.11 min
3885.1628
3825.35823824.7439
3810.35503722.7510
3707.4131
4251.3628
4324.2031
4340.55184411.9771
4442.8062
R-P-K-P-Q-Q-F-F-G-L-M
mPEGmPEG
123650 3700 3750 3800 3850 3900 3950 4000 4050 4100 4150 4200 4250 4300 4350 4400 4450
Faster separation with ion mobility p ymass spectrometry (IM-MS)
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IM-MS
trap IM transfer
LCsystem
p IM transfer
ion mobility region
Ions traveling through the IM cell are separated based on charge and collision cross-section, a function of size (mass) and shape.
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IM-MS combines separation and mass analysis in the same instrument, enabling top-down approaches
= complete characterization within the mass spectrometer.spectrometer.
Top-down approaches for large, labile, or not soluble / ionizable materials via ESI or ASAPsoluble / ionizable materials via ESI or ASAP
coupled with IM-MS / MS2.
ASAP = analysis of solids at atmospheric pressurey p p(mild thermal degradation in an atmospheric
pressure chemical ionization source)
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K. Guo et al., Macromol. Rapid Comm. 36 (2015) 1539-1552.
ASAP-IM-MS
Thermoplastic polyurethanes
dioldiisocyanate
diolchain
extender+ polyol
(small diol) (aromatic or aliphatic;linear or cyclic)
(polyether diol;polyester diol;
hard segments (m) soft segments (n)
linear or cyclic) polyester diol;PDMS diol)
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g ( ) g ( )
ASAP-IM-MS of a thermoplastic polyurethane (PU-1; elastollan)
250 oC10
aft tim
e (m
s)
b5drif
500 1000 1500 m/z
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ASAP-IM-MS of PU-1; low T (250 oC) desorbates
IM regiona BHT
MDI149 205220
292
277
0
MDIphthalate
120 150 180 210 240 270 m/z
250233
169
143 21
263
IM regionb
O
SiO
Si
OSi
nIdentification of
additives &
429503
74 Da
PDMSadditives &
diisocyanate
420 520 620 720 m/z
577651
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ASAP-IM-MS of a polyurethane PU-1; elastollan
450 oC
d10
s)
bc
5drift
tim
e (m
s
a
5
500 1000 1500 m/z
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ASAP-IM-MS of PU-1; high T (450 oC) products
250
32
180
194
208
224
314
4556268
250
412340
1 hard + n soft segment unitsMDI
72
IM regiona
72 72
120 220 320 420 520 m/z
1
106
1
322
430
536 56
4
592
556412
484
340 Da Da Da
hard segment
MDIBDOMDI
72-Da repeat unit and m/z values are consistent with poly(tetrahydrofuran)
soft segmenthard segment(n = 1-3)
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72 Da repeat unit and m/z values are consistent with poly(tetrahydrofuran), PTHF, as the soft segment and 1,4-butanediol, BDO, as the chain extender
(structures confirmed by MS2).
Verification of ASAP products from PU-1 by MS2
340.21 hard + 2 soft
i
Verification of ASAP products from PU-1 by MS
484.3
413 2 322 2
segment units
485.3322.2
413.2 322.2
MDI
413.2
469.
3
456.
3426.
3
384.
2
340.2
278.
2
368.
2
222.
122
4.1
232.
1 250.1
150 200 250 300 350 m/z400 450 500
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ASAP-IM-MS of PU-1; high T (450 oC) products
795 7IM region
b 772.5
09.6
0.6
*844.6
#
%
%
723.6
795.7
867.8
793.7865.8
675.
5
70
872.
6
860
879.
7
*
*#
#%%
$ $$739.6
811.7
680 720 760 800 840 m/z
*1 hard + n (5-7) soft segment units
# $soft
segment#soft
segment chains
$
%
segment chains
22Series with a 72-Da repeat unit
ASAP-IM-MS PU-1; high T (450 oC) products
IM regionc
636.5
592.6592.6
600 800 m/z
Irganox 1098
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ASAP-IM-MS of PU-1; high T (450 oC) products
4 3.6
1120.8IM regiond
656.
580
.3
700.
723
739.
6
772.
5
793.
679
5.7
7 7.7 916.6
1064.7
1176 8
[M-tBu]+one ester bond
hydrolyzed
700 800 900 1000 1100 1200m/z
68 7
811.
7
844.
6
865. 86
1008.6
1176.856Da
56Da56
Da
Irganox 1010
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H b id M t i lHybrid Materials
Hybrid materials usually consist of covalently linked y y ypeptides / proteins and synthetic polymers. Over the last decade, they have experienced increasing use in medicine and materials science, in a variety of consumer, industrial, and biomedical applications.pp
Hydrophilic polymers tend to stabilize peptide secondary & y p p y p p ytertiary structure, enabling function in biological & nonbiological environments.
S. Perrier et al., Polym. Rev. 51 (2011) 51:214–34; Xu et al., Annu. Rev. Phys. Chem. 64 (2013) 631–57
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Peptide-Polymer Hybrid Materials
Challenges in their characterization:
Peptide-polymer conjugates are difficult to crystallize for X-Peptide polymer conjugates are difficult to crystallize for Xray analysis.
Such hybrids cannot often be chromatographically purified for definitive NMR analysisfor definitive NMR analysis
Alternative solution: top-down MS, involving tandem MS (MS2) and ion mobility mass spectrometry (IM-MS).(MS ) and ion mobility mass spectrometry (IM MS).
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Elastin Mimetic Hybrid Copolymer
Hydrophilic domains (K and A rich) for crosslinkingElastin: extracellular protein
+Flexible hydrophobic domains
(V, G, and P rich) for coacervation
pproviding elasticity to soft tissues
(lungs, skin, arteries, etc.)
coacervation
+click rxn.
VPGVG–VPGVG
“VG2”(in hydrophobic
poly(acrylic acid)PAA
(pH-responsive &
X. Jia et al., Soft Matter 9 (2013) 1589-99
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(in hydrophobic elastin domains)
(pH responsive & functionalizable)
Hybrid material[PAA‒VG2][PAA VG2]m
+
PtBA VG2Cu(I) DMFCu(I) DMF
TFA [PtBA‒VG2]m
28[PAA‒VG2]m
y6y7y8
[VG2+Na+17]⁺c6
c7z10
1107.5
a₂a7
a₆
[VG2+Na+17]
c5
c8 c9 c10
VG2553.6
a]+
G2+
2Na]
• ⁺
7
c4a₂ a₆
17]• ⁺
²ESI MS2 (ETD)
VG2
[VG2+2Na]⁺²
1090 64 11.5 [V
G2+
N [VG
c₁₀• +
17
1113
.5
[VG₂+
Na+
z₁₀• *
ESI-MS2 (ETD)
VG2 sequence confirmed by MS2
1090.6
959.
4
0.2
101
y7
y
c₉• +
17
₈• +17
17
c₆•+17701.2
602.1c₅•+17
c₄•+17 4.4
855.
3700
640.
1
601.
1
y6 a₆
658.
1
739.
2 757.
2
798.
381
4.3
a₇y8 c₈
c₇• +c₄ +17
545.2 954
29
500 600 700 800 900 1000 m/z
8
Hybrid material / [PAA‒VG2]m
m/z
ESI-IM-MS2000
[PAA‒VG2]+3[PAA‒VG2]+2
NH4OAc (pH = 6.64)+ 1% MeOH
1000
[PAA VG2]
[PAA]+n
[PAA‒PtBA]+n
1.81 3.61 5.42 7.22 drift time (ms)[PAA‒PtBA‒VG2]+n
IM-MS removes chemical noise and separates the desired amphiphilic hybrid both by charge state as well as from incompletely hydrolyzed hybrid and unreacted polymer to enable conclusive compositional characterization.
30A. Alalwiat, S.E. Grieshaber, B.A. Paik, K.L. Kiick, X. Jia, and C. Wesdemiotis, Analyst, (2015) DOI: 10.1039/c5an01600b.
Hybrid material / [PAA‒VG2]m
783.043 807.074831.076
PAA14PAA15 PAA16
3+
ESI-IM-MS
780 790 800 810 820 830 m/z
07.0
7
804 07 80
831.
0
855.
04759.
783.
0
735.
03
711.
04
687.
03
63.0
1 879.
09
903.
10
927.
12
951.
14
[M+3H]3+
66 9
650 700 750 800 850 900 950 1000 m/z
ESI-IM-MS provides conclusive evidence for the formation of hybrid material with oneESI IM MS provides conclusive evidence for the formation of hybrid material with one constituent PAA–VG2 block, [PAA–VG2]1:
Multiple blocks?
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Hybrid material / [PAA‒VG2]m
2+
ESI-IM-MS
1030.06 1066.07PAA10
PAA11
30.0
6
6.07
07 38.1
0 1030 1040 1050 m/z1060 107010
3
994.
03
958.
02
922.
00
895.
97
106
1102
.
11
1210
.14
1246
.14
282.
14
318.
18
354.
211174
.12
[M+2H]2+
1 1 13
900 1000 1100 1200 1300 m/z
ESI IM MS id l i id f th f ti f h b id t i l ithESI-IM-MS provides conclusive evidence for the formation of hybrid material with one constituent PAA–VG2 block, [PAA–VG2]1:
Multiple blocks?
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H b id t i l / [PAA VG2]Hybrid material / [PAA‒VG2]m
ESI-IM-MSm/z
[PAA‒VG2]2+4
2000
[PAA‒VG2]+3[PAA‒VG2]+2
*
[PAA VG2]2
m/z 10301000 [PAA]+n
[PAA‒PtBA]+n
[PAA‒PtBA‒VG2]+n
m/z 1102
1.81 3.61 5.42 7.22 drift time (ms)
33
Hybrid material / [PAA‒VG2]m
ESI-IM-MS5.42
[PAA10‒VG2]1[M+2H]2+ m/z 1030
3.88
6.95[PAA10‒VG2]2[M+4H]4+
[PAA10+K]+
& [PAA24+Na+K]2+
0.00 2.50 5.00 7.50 10.00 drift time (ms)
IM-MS on mass-selected ions confirms the formation of a multiblock hybrid copolymer.
5.96
m/z 1102[PAA12‒VG2]1[M+2H]2+
[PAA12‒VG2]2
copolymer.
4.06 7.13
[ 12 ]2[M+4H]4+
[PAA11+K]+
& [PAA26+Na+K]2+
340.00 2.50 5.00 7.50 10.00 drift time (ms)
Hybrid material / [PAA‒VG2]mArchitecture?Architecture?
li ?
intramolecular azide click rxn.
linear ?
cyclic ?y
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Hybrid material / [PtBAn‒VG2]1ArchitectureArchitecture
ESI-IM-MS10
470
490
510
calcd., linear architecture78
10
calcd., linear
Drift time(ms)
410
430
450calcd., cyclic architecture
measured
Collisioncross-section
(Å2) n =
4
6
2+ i
calcd., cyclic
measured
350
370
390
900 1100 1300
Power (calcd., linear architecture)
Power (calcd., cyclic architecture)
m/z
2+ ions
900 1100 1300
With all chain lengths, the measured CCS matches the one calculated for the macrocyclic architecture, indicating that all possible 3+2 cycloadditions
36
have taken place (only triazole and no azide / alkyne functionalities).
Hybrid material / [PtBAn‒VG2]1ArchitectureArchitecture
ESI-IM-MS490
510
Collisioncross-section
(Å2) 450
470
490
calcd., linear architecture
calcd cyclic architecture
calcd., linear
calcd cyclic9
390
410
430calcd., cyclic architecture
measured
Power (calcd., linear architecture)
calcd., cyclic
measured3+ ions
5
67
m/z350
370
710 810 910
)
Power (calcd., cyclic architecture)n =
5
In the 3+ charge state, the composition is [PAA3-PtBAn-VG2 + 3H]3+. The agreement between measured CCS and calculated CCS for the macrocyclic architecture is again excellent (within 4%), confirming that only triazole functionalities are present
37
functionalities are present.
Hybrid material / [PAAn‒VG2]1ArchitectureArchitecture
ESI-IM-MS
ectio
n (Å
2 )
420
440
460
480
calcd., linear architecture
calcd., cyclic450
470
490calcd., linear architecture
calcd., cyclic
calcd., linear
calcd., cyclicectio
n (Å
2 )
n =10
12
1416
n =12
1415
1615
sion
cro
ss-s
e
360
380
400
420 calcd., cyclic architecture
measured
Power (calcd., linear architecture)390
410
430
, yarchitecture
measured
Power (calcd., linear architecture)
, y
measured
sion
cro
ss-s
e
78 10
Col
li
m/z320
340
900 1000 1100 1200
Power (calcd., cyclic architecture)
350
370
680 730 780 830
Power (calcd., cyclic architecture)C
olli
m/z
2+ ions 3+ ions
The measured CCSs are smaller then those of the macrocyclic architectures. This is attributed to extensive H-bonding between the COOH pendants and the VG2 amide groups, leading to compact conformations that were missed
38
the VG2 amide groups, leading to compact conformations that were missed in the simulations.
Multidimensional MS [interfaced separation & massanalysis methodologies] in polymer and materials sciencey g ] p y
Interactive LC is particularly useful for the separation of mixtures whose components differ significantly in polarity. On the other hand, IM separation is most effective for the separation of differently shaped polymers and ideally suitable for the analysis of labile / reactive / weakly bound polymers (e.g., hybrid materials & supramolecular polymers).
Slow thermal degradation interfaced with IM-MS leads to composition and structure insight on complex polymers that cannot be desorbed/ionized and are difficult to analyze otherwise.
Top-down MS with IM-MS and MS2 removes the need of high purity for structural characterization (as needed in XRD and NMR).
Collision cross-sections add a further dimension of structuralCollision cross-sections add a further dimension of structural differentiation & identification.
Significant improvement in the microstructure characterization of synthetic macromolecules
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synthetic macromolecules.
AcknowledgementsDr. Nilufer Erdem (Tubitak, Turkey) NSF( , y)Dr. Bryan Katzenmeyer (Valspar)Dr. Aleer M. Yol (FDA)Dr. Nadrah Alawani (Aramco)Dr. Xiaopeng Li (Texas State U)Ahlam Alalwiat
NSFOBRThe University of AkronGoJoLubrizolAhlam Alalwiat
Lydia CoolSelim Gerislioglu
GoodyearOmnova Solutions Foundation
Quirk - Cheng - Newkome -Pugh - Foster - Puskas - Jana -Weiss research groups
Dr. Xinqiao Jia (U Delaware)Dr. Sarah Grieshaber (U Delaware)Bradford Paik (U Delaware)
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Dale Chilhuly’s “Rocks”
M i D ti Evening - NighttimeMorning - Daytime
41
Rubber Companiesfounded in Akron
Cuyahoga ValleyCuyahoga Valley National Park
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