Automated direct monitoring of H/D exchange reactions for mapping conformational landscapes of proteins

Allis Chien, Sheila Jaswal Stanford University

What are Conformational Landscapes and why map them?

U Native protein (N) also populates an ensemble of non-N conformations N I1 In
less stable

I2

Funnel Landscape
U Entire equilibrium determines function

energy

I1 I2 N configuration

more stable

Why do we want to detect intermediates?

perturbed conditions

Amyloid fiber formation:

2microglobulin fibers. C. Eakin, Yale University

aggregation-prone intermediate is stabilized

Native State Hydrogen Exchange to map conformational landscapes of proteins

Accessible amide protons exchange with (deuterated) solvent with known kex

CO

N H

C kex

CO

N D

Protons buried in folded proteins exchange in unfolding events:

Closed

kop kcl

Open

kex

Exchanged

U I1 I2
HX protection pattern identifies N vs. I1 vs. I2 vs. U

Electrospray Ionization MS to monitor H/D Exchange

Counts Mass/charge (m/z)
protonated mass* deuterated mass non exchanging internal standard

protonated protein deuterated solvent

6+

monitor over time

*after immediate HX of surface protons

m*/z

(m + a) /z

(m + b) /z

(m + c) /z

(m + x)/z

t=0 what happens in between?

t= end

NanoMate sprays Actin in 5 mM ammonium acetate, pH 7

pH 7: native, folded state pH 5 initial pH 5 30 min

As the protein is denatured, it acquires more charges and shifts to lower m/z values.

pH 2.5

MRM:

5 H/D exchange rxns, 36 timepoints, 48hr

DP 6 106 205 237 267 299 503 983 1343 2995 EP EP EP EP EP EP EP EP EP EP EP 8 171 207 244 269 301 505 985 1345 2997 A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A 10 11 12 14 16 18 20 25 30 35 40 52 67 84 94 104 126 152 157 187 216 242 291 342 402 462 522 642 762 882 1002 1122 1242 1842 2202 2562 2922 B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B 40 41 42 44 46 48 50 55 60 65 70 81 90 100 114 130 144 161 190 220 250 280 340 400 460 520 640 760 880 1000 1120 1240 1480 1840 2200 2560 2920 C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C 72 73 74 76 78 80 82 87 92 97 102 112 122 132 147 162 177 192 222 252 282 312 372 432 492 552 672 792 912 1032 1152 1272 1512 1872 2232 2592 2952 D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D 109 110 111 113 115 117 119 124 129 134 138 149 159 169 184 199 214 229 259 289 319 349 409 469 529 589 709 829 949 1069 1189 1309 1549 1909 2269 2629 2989 E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E 135 136 137 139 141 143 145 150 155 160 165 175 185 195 210 225 240 255 285 315 345 375 435 495 555 615 735 855 975 1095 1215 1335 1575 1935 2295 2655 3015

timepts AP BP CP mix time 1 AP 0 BP 2 CP 4 DP 2 AP 22 BP 57 CP 62 DP 3 AP 179 BP 201 CP 203 DP 4 AP 231 BP 233 CP 235 DP 5 AP 261 BP 263 CP 265 DP 6 AP 293 BP 295 CP 297 DP 7 AP 497 BP 499 CP 501 DP 8 AP 977 BP 979 CP 981 DP 9 AP 1337 BP 1339 CP 1341 DP 10 AP 3017 BP 2991 CP 2993 DP 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36

A,B,C,D,E: 0,2,4,6,8% ACN; P: corresponding protonated controls

Hydrocarbon layering as an evaporation barrier

Density
(g/mL, 25C)

Bp (C) 174

vapor pressure 1mm Hg (16.5C)

Octane (C8) Decane (C10) Dodecane (C12) Tetradecane (C14)

0.70 0.73 0.75 0.76

125-127 11mm Hg (20C)

215-217 1mm Hg (47.8C) 252-254 1mm Hg (76.4C) 287 1mm Hg (105.3C)

Hexadecane (C16) 0.77

Sample pickup with hydrocarbon layering: dispense, then aspirate

AUI Method: get tip, mix, spray, (delay)

Spray parameters: 0.2 psi, 1.9 kV

tip#

AUI Step (spray)

Sample

Time (min)

Time btwn samples

Spray Time

Sampling Sequence:
750 AUI steps, 230 tips/nozzles
Change 96-tip trays at 3h, 16h, Re-synchronize timing
AUI Step (spray) Time (min)

tip#

Sample

1 2 3 4 5 6 7 8 9

3 6 9 12 15 3 6 9 12

AP BP CP DP EP A A A A

0 2 4 6 8 10 11 12 14

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52

Time btwn samples

3 6 9 12 15 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48 51 54 57 60 63 66 69 72 75 78 81 84 87 90 93 96 99 102 105 108 111 114 117 120 123 126 129 132 135 138 141

AP BP CP DP EP A A A A A A A AP A A A A B B B B B B B A B BP B CP B A B C C C C C C B C A C B C A C B C A DP D D

mix

mix

Spray Time

mix

2 2 2 2 2 1 1 2 2

0 2 4 6 8 10 11 12 14 16 18 20 22 25 30 35 38 40 41 42 44 46 48 50 52 55 57 60 62 65 67 70 72 73 74 76 78 80 81 82 84 87 90 92 94 97 100 102 104 106 109 110

1:15 1:15 1:15 1:15 1:15 0:30 0:30 0:45 1:15

2 2 2 2 2 1 1 2 2 2 2 2 3 5 5 3 2 1 1 2 2 2 2 2 3 2 3 2 3 2 3 2 1 1 2 2 2 1 1 2 3 3 2 2 3 3 2 2 2 3 1 1

1:15 1:15 1:15 1:15 1:15 0:30 0:30 0:45 1:15 1:15 1:15 1:15 2:15 4:15 4:15 2:15 1:15 0:30 0:30 0:45 1:15 1:15 1:15 1:15 2:15 1:15 2:15 1:15 2:15 1:15 2:15 1:15 0:30 0:30 0:45 1:15 1:15 0:30 0:30 0:45 2:15 2:15 1:15 1:15 2:15 2:15 1:15 1:15 1:15 2:15 0:30 0:30

Total Ion Chromatogram of Time Course

Total Ion Chromatogram of Time Course

0-360 min 136 spray events programmed, 121 successful: 89%

0-75 min

31 spray events programmed, 30 successful: 97%

HXMS reveals an intermediate populated by amyloidogenic precursor protein

fits to 3 species

shows population shift

fraction populated
0.1 min-1 0.05 min-1

tim e

0.05 min-1 0.1 min-1

m/z

time, min

Conclusion
Automated monitoring of reactions enabled by NanoMate & hydrocarbon layering z Future work
z z z z z z z z z

Refine Multiple Reaction Monitoring method Automate data analysis Stepwise AUI-like control in real time 386-tip trays Beckman Foundation Vincent & Stella Coates Foundation Dennis Price, Gary Schultz, Dave Bajkowski, Colleen van Pelt, Mike Lees

Looking forward to Thanks to