interfacing mass spectrometry with separation methods for ... · peptide-polymer hybrid materials...

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

1

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).

3

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.

5

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

9

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+


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


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)

13

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.

14

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)

15

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)

16

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

17

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

18

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

19

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)

20

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

21


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

23


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

24

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

25

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).

26

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

27

(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.


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?

31


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?

32

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


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

35

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


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


, 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

39

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)

40

Dale Chilhuly’s “Rocks”

M i D ti Evening - NighttimeMorning - Daytime

41

Rubber Companiesfounded in Akron

Cuyahoga ValleyCuyahoga Valley National Park

42

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