Application of Proteomic Methods to

Study the Nucleus

CBE555 Nanoscale Systems Biology
October 21st, 2014

Sangkyun Cho

Overview
1) Principles of proteomics & mass spectrometry.

2) Practical application of mass spectrometry to quantify

proteins

3) Application of mass spectrometry to understand the role

of lamins in the nucleus.

Protein Structure

Protein Structure

hydrophobic
hydrophilic

Energy

The Folding Funnel Model

Denatured

Natured

Conformational
Space

The Levinthal problem: to randomly search all possible conformations (>10100)

would take an unfeasibly long time. Protein folding typically takes s to ms.
Does protein folding in models and experiments reflect the conditions in the cell?

Proteins and the Proteome

Although we are 70 wt% water
Cytoplasm is ~ 20 wt% protein; nucleus is ~ 0.5 wt% protein
Average oligomerization state of proteins is ~ 4
Human genome has ~25,000 genes coding for protein
CYTOPLASM

NUCLEUS

D Goodsell

-Omics Science

Proteome: the set of proteins expressed in a given type of cells or an

organism at a given time under a particular set of conditions.
Proteomics: Large-scale study of proteins, particularly their structure &
function

Gerszten and Wang. Nature (2008) vol. 451 (7181) pp. 949-952

Principles of Mass Spectrometry

Recommended Paper

Aebersold and Mann. Nature (2003) vol. 422 (6928) pp. 198-207

Principles of Mass Spectrometry

Sample Preparation

Ionization: Electrospray

Electrospray Ionization
- Evaporation of solvent
- Continuous flow system
- Nano-electrospray runs at 25 nL/min

Ionization: MALDI

Matrix Assisted LASER Desorption/Ionization

- Discrete, dried sample spots

- Drop volume (before drying): 0.1 1.0 L
- LASER resolution: 10 m spot size

Sinapic Acid (example matrix)

MALDI Imaging

Principles of Mass Spectrometry

Mass Analysis: Sector Instrument

Magnetic Sector

Selection for momentum-to-charge ratio

Mass Analysis: Sector Instrument

Electrostatic Sector

Selection for kinetic-energy-to-charge ratio

Mass Analysis: Sector Instrument

Combination of magnetic and electric fields

allows selection by mass-to-charge ratio

BE (reverse) geometry system

Mass Analysis: Example of a Simple Mass Spectrum

Mass Analysis: Time-of-Flight (TOF)

Time of Flight Analyzer

=> Mass-to-charge ratio derived

from time of flight

Mass Analysis: TOF Instrument

Bruker Ultraflex III (like in UPenn chemistry dept)

Mass Analysis: Example of MALDI-TOF Data

Mass Analysis: Quadrupole

Oscillating radio-frequency electric fields between rods allow

selection by mass-to-charge ratio.
Quadrupole Ion Trap allows ions to be trapped in an oscillating
field and sequentially ejected, depending on mass-to-charge ratio.

Mass Analysis: Orbitrap MS

Orbitrap Resonance Chamber
Ions are trapped in an electric or magnetic field.
As they oscillate, they induce a current in a probe.
Fourier transform on this signal yields a set of resonant frequencies.
These frequencies relate to mass-to-charge ratios:
Ion path

External field

(where is comparable to
a spring constant for the
oscillation)
Oscillation

Mass analysis: Orbitrap Instrument

Autosampler

Quadrupole

Liquid
Chromatography
(LC) column
Orbitrap
ESI
injection

Thermo Scientific LTQ Orbitrap LC-MS/MS (like at the Wistar Institute)

Mass analysis: FTMS Instrument

Superconducting
Magnets.

Ion Detection

Measure a current from charged ions.

The number of ions may be small, so some form of signal amplification
may be required, e.g. electron multiplier:
Increasing potential

Ion
Electron cascade

Electrode

Continuous dynode detector

FTMS and Orbitrap instruments detect an induced current.

Tandem mass spectrometry MS/MS

Collision with an
inert gas allows
formation analysis of
fragment ions,
making analyte
identification easier.

TOF / collision / TOF

Quadrupole / collision / quadrupole

Tandem mass spectrometry MS/MS

x, y, z = C-terminus fragment ions

N-terminus

C-terminus

a, b, c = N-terminus fragment ions

Subscript indicates number of amino acids in the fragment.

Tandem mass spectrometry MS/MS

Modes of Operation
Product Ion Scan

Most common method for

LC-MS/MS proteomics

Selected/Multiple Reaction Monitoring (SRM & MRM)

Very selective analysis

Isotope Labeling as Means of Quantitation

Isotopes: atoms of the same element, but varying in the number of neutrons in the nucleus. They
therefore have very similar chemical properties, but differ in atomic mass e.g. carbon-12 vs. carbon-13

Condition 1

Condition 2

Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)

MS for proteomics

Han et al. Mass spectrometry for proteomics. Curr. Opin. Chem. Biol. (2008) vol. 12 (5) pp. 483-490

MS for proteomics

A range of techniques are routinely used, often in combination:

QIT = Quadrupole Ion Trap
LTQ = Linear Trap Quadrupole
Q/q = Quadrupole
TOF = Time of Flight
FTICR = Fourier Transform Ion Cyclotron Resonance

Han et al. Mass spectrometry for proteomics. Curr. Opin. Chem. Biol. (2008) vol. 12 (5) pp. 483-490

MS for proteomics

Mass Resolution: m2 / (m2-m1)

(where m2 and m1 are distinguishable masses)
Larger resolution is better
Mass Accuracy: ratio of the m/z measurement
error to the true m/z
ppm = parts per million
Sensitivity: atto (10-18) < femto (10-15) < pico (10-12)

Han et al. Mass spectrometry for proteomics. Curr. Opin. Chem. Biol. (2008) vol. 12 (5) pp. 483-490