In-line separation by capillary electrophoresis prior to top-down mass
spectrometric analysis enables sensitive characterization of proteoforms
111,21331Xuemei Han, Aaron Aslanian, Bryan Fonslow, Daniel McClatchy, Beth Graczyk, Trisha N. Davis, and John R. Yates III

1The Scripps Research Institute, La Jolla, CA, 2SCIEX, Brea, CA, 3University of Washington, Seattle, WAOverview

Top-down analysis of intact Dam1 complex proteins• Purpose – Improve the top-down analysis of proteoforms from protein complexes andwhole proteomes through efficient front-end CE separations• Methods – Capillary zone electrophoresis (CZE)-based separation using CESI-MS, theintegration of CE and ESI into a single dynamic process, and intact protein MS/MS• Results – CESI-MS facilitates separation, identification, and quantification of intact
proteoforms, including their post-translational modifications

Identification of proteoforms from a whole cell lysate

Figure 4: CESI-MS analysis of intact
Dam1 complex proteins. (A) Extracted
ion electropherogram (XIE) from 2.5 ng
injection and separation. (B) Intactprotein MS spectra from integration ofeach CE peak. Theoretical molecularweights (MW) and isoelectric points (pI)are displayed.

AIntroduction
MW – 32.1 kDa
pI - 4.6

Front-end separation has been a challenge for top down proteomics. Extensive multiple-stage separation
systems have been developed to catalogue intact proteins at the whole proteome level. However, functional
information might be obscured when the components of protein complexes and proteoforms are separated
into distinct fractions. To study specific protein complexes that are usually present at low levels in cells, wedeveloped a sensitive and efficient CE based top down platform that provides an in-depth characterizationof each subunit of a protein complex. Using this platform, we studied the yeast Dam1 complex which is vitalto chromosome segregation during mitosis. We also employed this method to analyze a fractionated lysate
from a moderately complex organism, Pyrococcus furiosus (Pfu).

100

MW – 34.9 kDapI - 8.3

Figure 8: Identification of disulfide bonds on 50S ribosomal protein Q8TZI4 in Pfu. Graphical fragmentation maps generated withProSightPC are shown for consideration of the (A) reduced protein form with a 4 Da mass matching error and the (B) non-reduced protein
form with two disulfide bonds, resulting in a 0.1 Da mass matching error.

8+

10,589.14 Da – 9+80
Relative Abundance

Figure 7: Characterization of hexose/maltose modified Pfu
protein Q8U442 (30S ribosomal protein S24e). (A) Orbitrap mass
spectrum of unmodified and modified forms of Q8U442. (B) HCD
fragmentation spectrum for precursor 987.8612+ and graphicalfragmentation map generated with ProteinMetrics’ Byonic.

Figure 8: Characterization of hypusine/deoxyhypusine
containing protein translation initiation factor 5A (IF5A) inPfu. Graphical fragmentation maps generated with ProSightPC
are shown for (A) deoxyhypusinated IF5A and (B) hypusinated
IF5A. This modification was not previously known in thiscommonly studied Pfu model organism.

MW – 10.5 kDapI - 4.2

1315.33

Mps1 treated

9070Conclusions

6050

9+

40

10+

30

11+
816.25854.67 877.50 907.17
850

To perform efficient front-end separation and ionization of intact proteins prior to mass spectrometry,
CESI-MS was performed using a SCIEX CESI 8000 coupled to a Thermo Elite MS. The CESIcapillary was covalently-coated using polyethylenimine to suppress protein-wall interactions.

www.PosterPresentations.com

MW – 38.4 kDa
pI - 9.2

Dam1-Mps1_PSDVB100_2ul_4SIM #276 RT: 68.63 AV: 1 NL: 1.36E6
F: ITMS + p ESI Full ms [550.00-1450.00]

0800

TEMPLATE DESIGN © 2008

MW – 10.8 kDa
pI - 5.3

Analysis of Dam1 complex in vitro phosphorylation

10Figure 3: Pfu cell lysate containing ~2050 proteins was fractionated and analyzed by CESI-MS.

MW – 18.9 kDa
pI - 4.8
MW – 8.1 kDa
pI - 7.9

MW – 27.5 kDa
pI - 10.0

20Figure 2: Schematic of CESI sprayer. A fusedsilica capillary is etched at the tip until porous
and inserted into a metal ESI needle. A
conductive liquid is pumped between the porous
tip and metal needle. When an ESI voltage isapplied, electrical contact is made with thebackground electrolyte (BGE) in the CESI
separation capillary by the conductive liquid andtransport of ions through the porous tip. The
inherent low flow rate from electroosmotic flow
(EOF), ~20 nL/min, has improved ionization
efficiency and reduced ion suppression,
beneficial for intact protein analysis.

BMW – 8.2 kDa
pI - 6.7

Figure 1: Representation of yeast Dam1 complex.
The Dam1 complex is a component of the outer yeast
kinetochore that has important functions in chromosome
segregation during mitosis. Phosphorylation of theprotein Dam1 by Mps1 kinase is required for coupling of
the kinetochore to the plus-ends of microtubules. In vitro
kinase assays and bottom-up proteomics werepreviously used to identify most phosphorylation sites.

Methods

MW – 15.1 kDa
pI - 4.1

900

950

P

1052.58
1060.58P

957.001169.33

1005.671000

P1325.421178.33
1282.50
1140.08

1074.501050

P1100m/z1150

1208.75 1229.25
1200

1250

1356.25 1384.00
1300

1350

1400

CID fragmentation
Figure 5: Interpretation of intact Dad1
protein MS and MS/MS spectra. The +9
charge state of phosphorylated Dad1 wasfragmented
andidentified
using
ProteinMetrics’ Byonic software.
Thephosphorylation was localized to a threonine
residue.

• CESI-MS is ideally suited for the sensitive top-down analysis of intact proteins• PEI coating of the CESI capillary facilitates efficient protein separations over a wide
range of isoelectric points and sizes• CZE separation of intact proteoforms allows for generation of high quality MS andMS/MS spectra for sequence and PTM identification• Localization and quantification of phosphorylation by CESI-MS can serve as a MSbased in vitro kinase assay at the intact protein level• Common and previously unknown proteoform post-translational modifications can befound using top-down proteomics with CESI-MSReferencesACB

DE

Figure 6: Detection, separation, and quantitation of phosphorylation stoichiometry by CESI-MS. Comparison of thephosphorylation states of untreated Dam1 complex subunits (A) Dad1p and (C) Duo1p, and Mps1p kinase-treated Dam1 complex
subunits (B) Dad1p and (D) Duo1p on selected charge states. The phosphorylated proteoforms were labeled with #P, where # is thenumber of phosphorylation states. (E) XIEs of the four major phosphorylation forms (1P, 2P, 3P, 4P at charge state 36+) for Duo1p
following in vitro Mps1p kinase treatment.

1. Han X, Wang Y, Aslanian A, Fonslow BR, Graczyk B, Davis TN, Yates JR 3rd, In-Line
Separation by Capillary Electrophoresis Prior to Analysis by Top-Down Mass Spectrometry
Enables Sensitive Characterization of Protein Complexes, J. Proteome Res., 2014, 13,6078−6086.
2. HanX, Wang Y, Aslanian A, Bern M, Lavallée-Adam M, Yates JR 3rd, Sheathless Capillary
Electrophoresis-Tandem Mass Spectrometry for Top-Down Characterization of Pyrococcus
furiosus Proteins on a Proteome Scale, Anal. Chem., 2014, 86 (22), 11006–11012.

AcknowledgementsThe presenters thank National Heart, Lung, and Blood Institute Proteomics Center Award
HHSN268201000035C, R01 MH067880, and 8P41GM103533-17 for funding this work.