Successful grant application extracts - DVS Sciences

brewerobstructionAI and Robotics

Nov 7, 2013 (4 years and 5 days ago)

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From a (successful) grant application by Tanner et al.

:

This is an extract from a successful grant that Scott wrote in late 2009. I do not know how
much of this has already been adapted directly into other people’s grant applications.


I
have omitted certain sections that are heavily biased towards the particul
ar governmental
group that sponsored the grant, and deleted certain particularly proprietary parts.


This
grant was reviewed by a few US referees, but I suspect that the wordmanship will not be
recognized widely: therefore, I authorize to use what you can
from this (but be careful that
the wording supports your particular direction).


This grant was constructed as a
collaboration of international laboratories that would evaluate and advise on the continuing
development of the mass cytometry technology.


Lay
person:

Mass cytometry is a transformational new technology

that allows the identification
and investigation of rare, diseased cells.

... is a
transformational technology

that
enables the detection and characterization of rare and
heterogeneous cell popu
lations, such as cancer stem cells, at the individual cell level.
It marries the
resolution, specificity and sensitivity of atomic mass spectrometry to the high throughput, single cell
analytical advantages currently provided by flow cytometry
.


Using

stab
le isotope (non
-
rad
ioactive metal)
tags,
many proteins can be identified and quantified simultaneously in single cells, yielding a biomarker
signature that distinguishes diseased cells at an early stage
when

therapies can be less aggressive and
more effect
ive
.


Technical:

The “mass cytometer” instrument addresses the analytical challenges that are
normally encountered using flow cytometry, by using “element
-
tagged”
immunological staining combined with atomic mass spectrometry
that provides

many more independent and quantitative detection channels. The technology has
...
been described as potentially transformational for medical research into the
genesis of disease and for clinical diagnostics. Mass cytometry provides, for the
first time,
the ability to simultaneously determine at least 30, and potentially as
many as 100, biomarkers in individual cells. This portends the ability to recognize
extremely rare diseased cells in patient’s samples, and offers a potentially
transformative step to
wards personalized healthcare characterized by early and
correct diagnosis that improves the efficacy of less
-
aggressive therapeutic
intervention.


More detail:

Mass cytometry is an innovative
new technology that facilitates massively multi
-
parameter
ass
ay of single cells and particles at high
throughput [
1
,
2
,
4
,
5
]. It

is poised to transform
medical research int
o the genesis of disease, enabling

rational drug discovery and
development, and offers a real opportunity for personalized clinical diagnosis a
nd prognosis.
The ability to simultaneously measure more than 30 (potentially up to 100) biomarkers should
allow the confident identification of rare cells (e.g., cancer stem cells) in complex patients’
samples, the subclassification of rare cell distribu
tions that indicate the progression of disease
and response to therapy, and provide an information
-
rich snapshot of a wide distribution of
protein translational modifications resulting from stimulation or suppression.

The technology addresses
applications
that are typically run by flow cytometer analyzers,

but
extends the capability to many simultaneous parameters. The detector is based on atomic mass
spectrometry
that provides exquisite resolution of many mass channels while bringing unique
quantitative ca
pabilities to the biological arena. The breakthrough realization lay in recognizing
the potential of element
-
tagged immunoassay linked with

atomic mass spectrometry
.

According
to this empowering method, antibodies are tagged with metal atoms (elements of

the periodic
table, or preferably their enriched stable isotopes) such that a given antibody is associated with
a unique isotope label. Incubation with a sample in the normal manner associates the isotope
tag with the target antigen. The tag is quantita
tively determined by Inductively Coupled Plasma
Mass Spectrometry (ICP
-
MS). The method can be extended to other affinity assays, including
those using oligonucleotides (genes), lectins, aptamers, etc.

ICP
-
MS is the
preferred means of determining the ele
mental composition, especially ultra
-
trace
components, of materials. It has found acceptance in various applications including
environmental (e.g., drinking, river, sea and waste water analyses), geological (e.g., trace
element patterning), clinical (e.g.,

determination of trace metals in blood, serum and urine) and
high purity materials (e.g., semiconductor reagents and components) analysis. With few
exceptions, it is a diagnostic tool unknown in the biological arena.

Briefly, a sample, most commonly an aerosol produced by nebulisation, is injected into a high
-

temperature plasma obtained by heating a flowing argon gas stream with radio frequency (RF)
energy. Under conditions approximating those at the surface of the su
n, the sample is promptly
vaporized,
atomis
ed and ionized as it flows through the plasma. High speed mass analysis
provides a "mass fingerprint" that identifies the elements contained in the sample. The particular
attributes of the method of note include:
wide linear dynamic range (up to 9 orders of
magnitude), exceptional sensitivity (sub
-
part per trillion, or attomole/microlitre, detection),
enormous abundance sensitivity (<10
-
4

overlap between adjacent isotopes), counting
-
statistics
-
limited precision, ab
solute quantification, and tolerance of concomitant
matrix [
7
].


The method is amenable to a variety of analytical formats for cellular analysis. The common
characteristic is the elemental labelling of the target analytes (proteins, nucleic acids,
polysa
ccharides; generically

biomarkers

) in the whole cell, or its lysate. A facile means for
immunological staining, using a metal chelating polymer or copolymer (MCP), has been
developed [
19
].
These polymers are designed with a functional group at one end
to enable the
polymer to be covalently attached to a monoclonal antibody (mAb) or other biological
macromolecule. In addition, the polymer has metal
-
chelating ligands to bind multiple copies of a
lanthanide (Ln) metal or Ln isotope. In this way, the MCP
-
la
beled mAb or biological molecule
can carry many copies of a Ln metal or isotope, or other metal ion, for a particular biological
application. Simultaneous analysis of many antigens is enabled by incubating a cell sample
with a cocktail of antibodies, each

type labeled with a different metal or isotope. After stringent
washing, or other separation, the different isotopes are indicative of their respective antigens (or
biomarkers). In each analytical format, the signal intensity of each isotope, when measu
red by
ICP
-
MS, provides quantitative information about the number of copies of each antigen present.


Different analytical challenges can then be addressed by:

{I’ve deleted 4 or 5 rather proprietary
applications...}

(1)

Solution (or bulk) analysis: the stain
ed sample is digested in acid to form a
homogeneous solution that can be analyzed by conventional ICP
-
MS [
8
]. This quantifies
the biomarker signature averaged over the cell ensemble. It may have diagnostic value
when the patient is in the blast stage and

the average signature over the sample is
sufficient as a prognostic indicator. It also answers the need for a multi
-
parameter
ELISA assay. In the present proposal, the method will be frequently used to
characterize, qualify and quantify binding characte
ristics of different tagging constructs
and affinity materials
[
8
,
9
,
10
,
11
].

(2)

Mass cytometry: a suspension of whole stained cells is
nebulis
ed in a manner to
stochastically introduce individual cells into the ICP whereupon a full multi
-
parameter
analysis
of each cell is effected. Since each cell generates a transient signal of only
200
-
400 microseconds duration, a purpose
-
specific instrument configuration that
provides high
-
speed data collection and interpretation is demanded. The method can be
considere
d a massively multi
-
parameter analog of fluorescent flow cytometry. It offers
the unique ability to distinguish, identify and interrogate rare cells in complex (patients’)
samples [
1
].

(3)

Multiplexed Mass Cytometry: while mass cytometry is inherently multi
-
p
arameter, it can
simultaneously be used for multiplexed analysis, wherein different cell samples are
distinguished through uptake of a metal
-
labeling solution or endocytosis of metal
-
encoded beads. An admixture of samples that have been separately encoded

and
stained allows high
-
throughput assay with deconvolution via the encoding signals.


References for the above section:

REFERENCES:

1
.
Mass Cytometry
: A Novel Technique for Real Time Single Cell Multi
-
target Immunoassay
based on Inductively Coupled Plasma Time
-
of
-
Flight Mass
-
Spectrometry,
D. R. Bandura,
V.I. Baranov, O.I. Ornatsky, A. Antonov, R. Kinach, X. Lou, S. Pavlov, S. Vorobiev, J. E.
Dick and
S.D. Tanner,
Analytical Chemistry

(
accepted

2009 | doi: 10.1021/ac901049w
)

2
.
Elemental Analysis of Tagged Biologically Active Materials,

Vladimir I. Baranov,
Scott D. Tanner, Dmitry R. Bandura and Zoe Quinn, US patent number
7,135,296
(issued November 14,

2006).

3
.
Method and Apparatus for Flow Cytometry Linked with Elemental Analysis
,

Vladimir I. Baranov, Dmitry R. Bandura and Scott D. Tanner. US patent
number 7,479,630 (issued January 20, 2009).

4
.

Flow Cytometer with ICP
-
MS Detection for Massively Multi
plexed Single Cell
Biomarker Assay
, S.D. Tanner, D.R. Bandura, O. Ornatsky, V.I. Baranov, M.
Nitz and M.A. Winnik,
Pure and Applied Chemistry
,
80
, 2627
-
2641 (2008).

5
.
Multiplex Bio
-
Assay with Inductively Coupled Plasma Mass Spectrometry: Towards a
Massively Multivariate Single Cell Technology
, S.D. Tanner, O. Ornatsky, D.R. Bandura
and V.I. Baranov,
Spectrochimica Acta Part B
,
62
, 188
-
195
(2007).

6
.
A Sensitive and Quantitative Element
-
Tagged Immunoassay with ICP
-
MS
Detection,
V.I. Baranov, Z. Quinn
, D.R. Bandura and S.D. Tanner,
Analytical
Chemistry
,
74
,1629
-
1636 (2002).

7
.
The Potential for Elemental Analysis in Biotechnology,
V.I. Baranov, Z.A. Quinn
D.R. Bandura and S.D. Tanner,
Journal of Analytical Atomic Spectrometry
,
17
,
1148
-
1152 (2002).

8
.
Development of Analytical Methods for Multiplex Bio
-
assay with Inductively
Coupled Plasma Mass Spectrometry,
O.I. Ornatsky
,
R. Kinach
,
D.R. Bandura
,
X. Lou
,
S.D. Tanner
,
V.I. Baranov
,
M. Nitz and M.A. Winnik,
Journal of
Analytical Atomic Spectrometry
,
23
,
463
-
469 (2008).

9
.
Multiple Cellular Antigen Detection by ICP
-
MS,
O. Ornatsky, V.I. Baranov, D.R.
Bandura, S.D. Tanner, and J. Dick,
Journal of Immunological Methods
,
308
,
68
-
76 (2006).

10
.
Element
-
tagged immunoassay with ICP
-
MS detection: evaluation and
comparison to conventional Immunoassays
, E. Razumienko, O. Ornatsky, R.
Kinach, M. Milyavsky, E. Lechman,

M.A. Winnik and S.D. Tanner,
Journal of
Immunological Methods

,

336
, 56
-
63 (2008).

11
.
Lectins Conjugated to Lanthanide
-
Chelating Polymers
, M.D. Leipo
ld, I. Herrera,
O. Ornatsky, V. Baranov and M. Nitz,

Journal of Proteome Research,

8
, 443
-
449 (2009).

12
. “
Combination of immunohistochemistry and laser ablation ICP mass
spectrometry for imaging of cancer biomarkers
”,
J. Seuma, J. Bunch, A. Cox, C.
McLeod, J. Bell and C. Murray
,
Proteomics
,
8
, 3775
-
3784 (2008).


13
.

Labelling of proteins by use of iodination and detection by ICP
-
MS
”, N. Jakubowski, J.
Messerschmidt, M. Garijo Anorbe, L. Waentig, H. Hayen, P. H. Roos,
Journal of Analytical
Atomic Spe
ctrometry
,
23
, 1487 (2008).

14
.

Labelling of proteins with 2
-
(4
-
isothiocyanatobenzyl)
-
1,4,7,10
-
tetraazacyclododecane
-
1,4,7,10
-
tetraacetic acid and lanthanides and detection by ICP
-
MS
”, N. Jakubowski, L.
Waentig, H. Hayen, A. Venkatachalam, A. von Bohlen, P. H. Roos, A. Manz,
Journal of
Analytical Atomic Spectrometry,

23
, 1497
(2008)
.

15
.

Labelling of antibodies and detection by laser ablation inductively coupled
plasma mass spectrome
try. PART III. Optimization of antibody labelling for
application in a Western blot procedure
”, L. Waentig, P.H. Roos and N.
Jakubowski,
Journal of Analytical Atomic Spectrometry
,
24
, 924
-
933 (2009).

16
.
D. L. Costantini, C. Chan, Z. Cai, K. A. Vallis, and

R. M. Reilly,
J. Nucl. Med.
,
2007,
48
, 1357.

17
.

D. L. Costantini, M. Hu, and R. M. Reilly,
Cancer Biother. Radiopharm.
, 2008,
23
, 3.

18
.
Messenger RNA Detection in Leukemia Cell Lines by Novel Metal
-
Tagged in situ
Hybridization Using Inductively
Coupled Plasma Mass Spectrometry
,
O.
Ornatsky, V.I. Baranov, D.R. Bandura, S.D. Tanner, and J. Dick,
Translational
Oncogenomics

1,

1
-
9 (2006).

19
.
Polymer
-
Based Elemental Tags for Sensitive Bioassay,
X. Lou, G. Zhang, I.
Herrera, R. Kinach, O. Ornatsky, V. Baranov, M. Nitz, M.

A. Winnik,
Angewandte Chemie International Edition
,
46
, 6111
-
6114 (2007).

20
.
Antibody
-
Dendrimer Conjugates: The Number, Not the Size of the Dendrimers, Determines
the Immunoreac
tivity,

C. Wängler, G. Moldenhauer, M. Eisenhut, U. Haberkorn, and W.
Mier,
Bioconjugate Chem.
,
19
, 813

820 (2008).

21
. “
Controlled Synthesis and Water Dispersibility of Hexagonal Phase NaGdF4:Ho3
+
/Yb3
+
Nanoparticles
”,

R. Naccache, F. Vetrone, V. Mahalingam, L. A. Cuccia, J. A. Capobianco,
Chemistry of Materials
,
21
, 717 (2009).

22
.


From Trifluoroacetate Complex Precursors to Monodisperse Rare
-
Earth Fluoride and
Oxyfluoride Nanocrystals with Diverse Shapes through Cont
rolled Fluorination in Solution
Phase
”,

X. Sun, Y. W. Zhang, Y. P. Du, Z. G. Yan, R. Si, L. P. You, C. H. Yan,
Chemistry
-
a European Journal
,
13
, 2320 (2007).

23
. “
Biocompatible Hybrid Nanogels
”, A. Pich
, F. Zhang, L. Shen. S. Berger, O. Ornatsky, V.
Baranov, M. A. Winnik,
SMALL
,

4
,

2171
-
2175

(2008).

24
. “
Lanthanide
-
Containing Polymer Microspheres by Multiple
-
Stage Dispersion Polymerization

for

Highly Multiplexed Bioassays
”,

A. I. Abdelrahman
, S. Dai, S. C. Thickett, O. Ornatsky,
D. Bandura, V. Baranov and M. A. Winnik J. Am. Chem. Soc, (submitted for publication,
May 2009).

25
. “
A metal
-
coded affinity tag approach to quantitative proteomics
”, R. Ahrends, S. Pieper, A.
Kühn, H. Weisshoff, M.
Hamester, T. Lindemann, C. Scheler, K. Lehmann, K. Taubner,
M.W. Linscheid,
Mol. Cell. Proteomics
,
6
,

1907 (2007).

26
. “
Causal protein
-
signaling networks derived from multiparameter single
-
cell data”,

K. Sachs,
O. Perez, D. Pe'er, D.A. Lauffenburger
, and G. Nolan,
Science

308
, 523
-
529 (2005).

27
.

"
Learning signaling network structures from sparsely distributed data
", K.
Sachs, S. Itani, J. Carlisle, G. P. Nolan, D. Pe'er and Douglas A. Lauffenburger,
RECOMB proceedings and Journal of Computational Bi
ology

16
(2), 201
-
20
(2009).

28
. “
Learning cyclic signaling pathway structures while minimizing data
requirements
“,K. Sachs, S. Itani, J. Fitzgerald, L. Wille, B. Schoeberl, M.A.
Dahleh and G.P. Nolan,
Proceedings of the Pacific Symposium on
Biocomputing
,
2009.

29
. “
Formalism and structure learning for cyclic networks
”, S. Itani, M.
Ohannessian, K. Sachs, G. P. Nolan and M. A. Dahleh,
Journal of Machine
Learning Research (JMLR)
, (in revision).

30
. “
Characterization of patient specific signaling via augmenta
tion of Bayesian networks with
disease and patient state nodes
”, K. Sachs, A. J. Gentles, R. Youland, S. Itani, J. Irish, G.
P. Nolan and S. K. Plevritis,
31st Annual International Conference of the IEEE Engineering
in Medicine and Biology Society

(accepte
d, to appear)

31
.

Seventeen
-
colour flow cytometry: unravelling the immune system

, S.P. Perfetto,
P.K.
Chattopadhyay and M. Roederer,

Nat Rev Immunol

4
, 648

55 (2004).



Figures for above section:



FIGURES:




Figure 1:
Left: emission spectra for 8 commonly use fluorophores. Right: mass spectra of 30
enriched stable lanthanide isotopes.



Figure 2:
Experimental protocol for tagging antibodies with metal
-
chelating
polymers. The antibody of interest is subjected to select
ive reduction of

S
-
S
-
groups to produce reactive
-
SH groups, which are reacted with the terminal
maleimide groups of a polymer bearing metal
-
chelating ligands along its backbone.
The polymer
-
bearing antibodies are purified, treated with a given lanthanide
ion,
and then purified again. Each type of antibody is labeled with a different element.
(from reference
19
).



Figure 3: Schematic of the prototype Mass Cytometer, comprising a novel configuration of ICP
-
TOF
-
MS (from reference
1
).




Figure 4:
A PBMC
(Peripheral Blood Mono
nuclear

Cell) sample was probed with antibodies
against 15 cell surface proteins (antigens), each antibody being labeled with a different stable
isotope, and the DNA was stained with an Ir intercalator. The data is displayed here in
the 16
2
-
1= N two
-
dimensional plots that are theformat of conventional flow cytometry (using FlowJo
software).







Figure 5:
Polar diagrams of median intensity values for surface antigens measured
using metal
-
tagged antibodies on leukemia patient sample
s, monoblastic M5
AML(A) and monocytic M5 AML (B). The monoblastic phenotype (A) shows high
CD33 and HLA
-
DR and low CD34, CD13, CD14, and CD64 expression levels. The
more differentiated monocytic M5 (B) type displays increased CD13, CD14, and
CD64 and lowe
r HLA
-
DR levels. Data were collected on the mass cytometer and
processed with FlowJo software. The typical population size used for averaging was
15 000
-
20 000 cells. Each of the 22 axes represents an antibody (or contrast
reagent, CR, or Ir
-
DNA intercalat
or) measured by detecting the isotopic tags
indicated in Table 2 per individual cell event. Samples were generously provided by
the Quebec Leukemia Cell Bank. (from reference
1
).