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Proteomics, small molecules and metablomics 03
Membrane drug transporter quantification 09
The 60th annual conference on Mass Spectrometry
and Allied Topics 14
Anthony Bristow, AstraZeneca, poses the questions for our
Mass Spectrometry Leaders Roundtable 15
IN-DEPTH FOCUS
SPONSORS
European Pharmaceutical Reviewwww.europeanpharmaceuticalreview.com Volume 17 | Issue 2 | 2012
01
MASS SPECTROMETRY
mass spec lead page SUPP_Layout 1 25/04/2012 14:01 Page 1
waters_Layout 1 22/03/2012 09:52 Page 1
A major breakthrough was achieved with the
development of two soft ionisation tech-
niques, namely matrix-assisted laser desorption
ionisation (MALDI)3 by Karas and Hillenkamp
and electrospray ionisation (ESI)4 by J. Fenn
(Nobel Prize Winner, 2002). These techniques
opened the door to analysing macromolecules
by MS.
The combination of gas chromatography
(GC) and MS was introduced in 19595. Liquid
chromatography coupling MS (LC-MS), however,
appeared much later6 and its interface with EI
was limited. This situation changed after the
development of other ionisation methods,
especially ESI. Today ultra-pressure liquid
chromatography (UPLC) is broadly used due to
its higher speed, resolution and sensitivity.
CompositionA mass spectrometer is composed of three
fundamental parts: ion source, mass analyser
and detector. It also commonly includes sample
introduction devices (GC or LC) and a data
processing system.
Ion sourceOur ability to analyse compounds depends on
the ability to generate ions from them. Many
types of ion sources have been developed and
the most widely used nowadays are ESI or
MALDI (Figure 1).
ESI7 is performed by applying an electric
field to a liquid passing through a capillary tube
whereby the field induces charge accumulation
at the liquid surface, ending with the formation
of highly charged droplets. These droplets then
pass through a warm gas until complete solvent
evaporation. ESI is suitable for the analysis of
both small and macromolecules and the
technique produces multiple charged ions
which allow molecular weight (MW) determi -
nation in analysers with a mass range limit as
low as 2000 Da4.
Mass spectrometry (MS) is a well-established analytical tool. Approximately 100years ago, J.J. Thomson constructed the first mass spectrometer to quantitativelymeasure the mass and charge of cathode rays (Nobel Prize Winner, 1906)1. The firstcommercial instrument was built in the early 1940s using electron impact ionisation(EI) and magnetic deflection. Other mass analysers, such as time of flight (TOF) andion cyclotron resonance (ICR) appeared 10 years later. Today, the most widely usedequipment includes quadrupole (Q) analysers2 (awarded the Nobel Prize in 1989),quadrupole ion traps (IT) and TOF.
MS IN DRUGDISCOVERY
Ana Rita Angelino and Min YangDepartment of Pharmaceutical and Biological Chemistry, UCL School of Pharmacy
European Pharmaceutical Reviewwww.europeanpharmaceuticalreview.com Volume 17 | Issue 2 | 2012
03
FIGURE 1 Ionisation methods: a) ESI; b) MALDI
yang SUPP_Layout 1 25/04/2012 14:02 Page 1
MALDI3 produces intact gas-phase ions
from a broad range of large, non-volatile and
thermally labile compounds, such as proteins,
oligonucleotides and polymers. The sample is
prepared by mixing an analyte with small
organic molecules (the matrix), which have a
strong absorption at the laser wavelength. Dried
mixture portions are ablated by a laser, which
induces sublimation of matrix crystals and
entrain intact analytes in the expanding matrix
plume8. The MALDI technique is independent of
absorption properties and the size of the
analysed compound and usefully it allows
femtomole range detection of proteins with
MW of up to 300 kDa9.
Mass analyserThe mass analyser is the component where ions
are separated according to their m/z values and
common formats are: Quadrupole (Q), Time of
Flight (TOF) and Ion Trap (IT).
The Q analyser’s basic principle was first
described by Nobel Prize Winner W. Paul in the
1950s10. The equipment is composed of four
parallel electrical rods and a direct current
potential is applied to two rods and the
remaining two are linked to an alternative radio-
frequency potential. Ions formed are pulsed
towards the compartment by the electric field
and separated by their m/z ratios. Q mass
spectrometers are commonly combined with
either GC or LC as a simple high throughput
screening (HTS) system. They can also be placed
in tandem to perform fragmentation studies.
The simple TOF methodology was first
described in the middle of the 20th century11,
but remained without significant visibility until
the 1990s12. It is based on the free flight of ions
through a known distance and the m/z value is
directly proportional to the time required for the
ions to traverse the length of the flight tube.
TOF has a broader MS detection range (up to
1.5 MDa) and is widely used in tandem MS.
The IT analyser was developed in parallel
with the Q technology by Paul’s group10. In ion
trap, an electrostatic ion gate pulses to inject
ions into the analyser. The pulsing action
differentiates IT equipment from beam
instruments, where ions continually enter the
analyser. Ions are then captured for a certain
time in the trap compartment. Due to its
trapping nature, IT analysers are especially
suited to perform multiple stages of MS (MSn)
experiments in structural elucidation studies13.
They are robust, sensitive and relatively
inexpensive, however they suffer from low
accuracy. They are usually coupled with ESI and
MALDI as well as with LC techniques.
In 2000, A. Makarov invented the Orbitrap
(OrbT)14. This equipment is considered a
modified IT that uses a quadrologarithmic
electrostatic potential, created between the
axially symmetric electrodes, to trap the ions.
Stable ion trajectories combine rotation around
a central electrode with harmonic oscillations.
The ions frequency, characteristic of their m/z
values, is detected and converted to a spectrum
using a fast Fourier transform (FT), similar to that
used in FT-ICR.
OrbT was considered a potential alternative
for FT-ICR, due to its high resolving power, good
internal mass accuracy and high space charge
capacity. Nowadays, OrbT is commonly used in
MS/MS experiments, combined with diverse
types of mass analysers.
Tandem Mass Spectrometry (MS/MS)MS/MS strategy was developed to overcome the
problem associated with the little fragmentation
of peptide ions created by soft ionisation
techniques. For this purpose, a single Q analyser
can be attached to other analysers, such as an
additional Q (triple quadrupole, QQQ) or a TOF
(Q-TOF). Other common MS/MS equipment
includes TOF-TOF, LTQ-OrbT and ion mobility
separation MS (SYNAPT).
Generically, MS/MS is based on a precursor
ion that is mass-selected in the first stage of the
analysis. In the second stage, this ion reacts
forming charged fragment products that will be
further analysed in the third stage (Figure 2).
The QQQ instrumental approach was
introduced in the late 1970s15 and consists of
three quadrupole compartments connected
online. The first Q acts to scan the precursor ions.
A selected ion is isolated and undergoes
dissociation by a process termed collision-
induced dissociation (CID) in the second Q. The
third Q then behaves as the standard analyser.
The most useful functions of QQQ are: precursor
ion scan, product ion scan, neutral loss scan
(used to study post-translational modifications,
PTMs) and multi-reaction monitoring (MRM,
used in pharmacokinetics, PK).
The Q-TOF instrument was first described in
199616 and combines the scanning capabilities
of a Q system and the resolving power of a TOF
analyser. It can provide high quality, informative,
simple, one-stage MS and MS/MS data.
Mass spectrometry applications in drug discoveryMS technology has spread its application field to
every discipline within the life and health
sciences. In this section, we summarise the most
common applications that MS-based methods
currently play in drug discovery as well as the
latest method developments.
Drug target identification – proteomicsProteomics is the large-scale study of proteins,
particularly of their structures and functions. The
full genome map and mRNA study cannot
reflect the real intracellular protein level17.
Proteins are subject to PTMs that modify their
function or location and some of them
demonstrate abnormal expression levels in
diseased tissues. MS is the method of choice to
identify complex protein samples either as
drug targets or as biomarkers18. Identification
can be carried out via peptide mass finger -
printing (PMF) or using MS/MS ions to search
against databases. A number of techniques
can be used, including bottom-up and top-
down approaches.
The bottom-up approach presents the
original concept of expression proteomics. It can
be defined either as the attempt to identify all
expressed proteins present in cells, tissues and
organisms or the differential analysis of
biological systems reacting upon external
stimuli or under specific disease conditions.
Bottom-up relies on chemical or proteolytic
European Pharmaceutical ReviewVolume 17 | Issue 2 | 2012
04
MASS SPECTROMETRY IN-DEPTH FOCUS
FIGURE 2Tandem mass spectrometry
“ MS is the method of choice to identify complex protein
samples either as drug targets or as biomarkers ”
yang SUPP_Layout 1 25/04/2012 14:02 Page 2
protea_Layout 1 05/04/2012 09:34 Page 1
cleavage of a protein into peptides prior to any
MS analysis19. It is generally initiated by one or
two-dimensional gel electrophoresis (2-DE) as
the standard separation techniques. In 2-DE,
proteins are first separated according to their
isoelectric points (PIs) and secondly by their MW.
The conceptual alternative to the bottom-
up approach is the top-down approach, a term
popularised by McLaffertty’s group20. It involves
the gas-phase dissociation of intact proteins
with ions formed being subjected to MS/MS
analysis. Recently, two new dissociation
techniques were introduced: electron capture
dissociation (ECD)21 and electron transfer
dissociation (ETD)22. They provide more uniform
molecule dissociation than conventional CID
(for example, ETD can selectively break
glycosidic bonds, which is very useful in
glycosylation studies). Yates III developed a
powerful tool known as multidimensional
protein identification technology (MudPIT)
which can identify 1,000 – 2,000 proteins in a
single fraction23.
Quantitative proteomicsClassical quantitative proteomics includes gel
image comparison and/or difference in-gel
electrophoresis (DIGE) using fluorescent dyes24.
Recent developments employ differential stable
isotope labelling to create specific mass tags
that can be recognised by MS and, furthermore,
quantified. These include: metabolically
added isotope labelling by amino acids
(SILAC)25, commonly 13C6-arginine and 13C6-lysine26 than 15N27; chemical methods as
an isobaric tag for relative and absolute
quantitation (iTRAQ)28, isotope-coded protein
label (ICPL)29 and tandem mass tags (TMT)30;
enzymatically labelled with 18O during protein
digestion31 and label free quantification32.
Protein / ligand interactionWith the development of soft ionisation
techniques, MS has been used to characterise
target-ligand interactions. Experiments focus on
the ligand, target-ligand complex or ligand
binding site, in either a qualitative (HTS) or a
quantitative way (characterisation). Two
advantages have been demonstrated: 1) the
ability to monitor interacting partners without
labelling them and 2) the ability to identify
structurally unknown hits.
The pharmaceutical industry currently aims
to develop a HTS method to find potential drug
candidates in large compound libraries. Few
methods have been developed to study the
interactions via ligands, such as gel permeation
chromatography (GPC) spin columns33, the
automated ligand identification system (ALIS)34
and ultrafiltration35. The fundamental concept is
to mix library compounds in a column or a
membrane and to try and separate those bound
with unbound compounds, identifying those
bound by MS. These methods generally
work for compounds with Kd<10μM. A con -
tinuous-flow system, as a chip-based device,
requires a pump, injectors, mixtures, reactors
and an MS detector and presents several
advantages36,37. Frontal affinity chromatography
(FAC)38 is a biophysical method to discover
and characterise molecular interactions in
a flow-based system. There are several
analytical methods from the robust single-
compound characterisation to HTS of over
1,000 compounds per run. Compounds are in a
dynamic equilibrium with the immobilised
target. Unbound and weakly bound compounds
are eluted earlier than the others. Mass-
specificity in detection allows identification of
active compounds and these methods not only
provide a screening result for a lead candidate,
but can also be used to determine dissociation
constants (Kd).
Interactions via a protein-ligand complex
have been studied using a tethering (fragment-
based drug discovery) technique39 and ligand
titration binding40. Protein-ligand interactions in
solution by hydrogen/deuterium exchange
(PLIMSTEX)41 and hydrogen/deuterium ex -
change mass spectrometry (DEMS)42 can also be
used to study the binding site.
Quantification of small moleculesPharmacokinetics (PK) studies the fate of
administered drugs in a living organism and is
divided into several processes known as ADME
(absorption, distribution, metabolism and
excretion). LC-MS, especially QQQ MS/MS, is
commonly used in PK due to its advantages:
1) the LC system easily separates the complex
matrix (plasma or urine) and 2) it provides a
unique MRM facility.
In MRM43, an ion of interest (the precursor) is
selected in Q1, then induced to fragment in the
collision cell (Q2). The fragment ion signal is then
monitored over the chromatographic elution
time. Selectivity, resulting from the two filtering
European Pharmaceutical ReviewVolume 17 | Issue 2 | 2012
06
MASS SPECTROMETRY IN-DEPTH FOCUS
1. Thomson, J. J. Proc. R. Soc. 1913, 89, 1
2. Finnigan, R. E. Anal. Chem. 1994, 66, 969A
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10. (a) Paul, W.; Steinwedel, H. Zeitschrift fürNaturforschung 1953, 8A, 448(b) Paul, W. Angew.Chem. Int. Ed. Engl. 1990, 29, 739
11. Stephens, W. E. Phys. Rev. 1946, 69, 691
12. Brown, R. S.; Lennon, J. J. Anal. Chem. 1995, 67, 1998
13. Patel, S. M.; Fuente, M.; Ke, S.; Guimaraes, A.; Oliyide, A.O.; Ji, X.; Stapleton, P.; Osbourn, A.; Pan, Y.; Bowles, D. J.;Davis, B. G.; Schatzlein, A.; Yang, M. Chem. Commun.2011, 47, 10569
14. Makarov, A. A. Anal. Chem. 2000, 72, 1156
15. Yost, R. A.; Enke, C. G. J. Am. Chem. Soc. 1978, 100, 2274
16. Morris, H. R.; Paxton, T.; Dell, A.; Langhorne, J.; Berg, M.;Bordoli, R. S.; Hoyes, J.; Bateman, R. H. Rapid Comm.Mass Spectrom. 1996, 10, 889
17. (a) Rogers, S.; Girolami, M.; Kolch, W.; Waters, K. M.; Liu,T.; Thrall, B.; Wiley, H. S. Bioinformatics 2008, 24,2894(b) Dhingraa, V.; Gupta, M.; Andacht, T.; Fu, Z. F. Int.J. Pharm. 2005, 299, 1
18. Wong, S. C. C.; Chan, C. M. L.; Ma, B. B. Y.; Lam, M. Y. Y.;Choi, G. C. G.; Au, T. C. C.; Chan, A. S. K.; Chan, A. T. C.Expert Rev. Proteomics 2009, 6, 123
19. Wittmann-Liebold, B.; Graack, H. R.; Pohl, T. Proteomics2006, 6, 4688
20. Kelleher, N. L.; Lin, H. Y.; Valaskovic, G. A.; Aaserud, D. J.;Fridriksson, E. K.; McLafferty, F. W. J. Am. Chem. Soc.1999, 121, 806
21. (a) Zubarev, R. A.; Horn, D. M.; Fridriksson, E. K.;Kelleher, N. L.; Kruger, N. A.; Lewis, M. A.; Carpenter, B.K.; McLafferty, F. W. Anal. Chem. 2000, 72, 563(b)Cooper, H. J.; Håkansson, K.; Marshall, A. G. MassSpectrom. Rev. 2005, 24, 201
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REFERENCES
“ With the development of softionisation techniques, MS has
been used to characterise target-ligand interactions ”
“ The pharmaceutical industrycurrently aims to develop a
HTS method to find potential drug candidates in large
compound libraries ”
yang SUPP_Layout 1 25/04/2012 14:02 Page 3
stages, combined with the high duty cycle,
results in quantitative analysis with unmatched
sensitivity and specificity.
Yang et al. have developed an MS-based
HTS method which can be used to quantitatively
analyse multi-substrate enzyme full kinetics as
well as the substrate specificity44. This method
relies on a single Q system with selected ion
monitoring (SIM) and an internal standard (IS) is
crucial for quantification of this type as it
calibrates the ionisation degree of the products.
A double injection approach has been applied
to analyse IS and the reaction mixture
simultaneously without interference between
each other.
Other approachesMS has also been used in metabolomics to
identify and quantify intracellular and
extracellular metabolites with a MW lower than
1000 Da45. This provides information on
unregulated pathways and drug metabolism.
Additionally, imaging MS (IMS) can be used to
analyse protein expression via secondary ion MS
(SIMS) and MALDI imaging46. This combines
chemical specificity, MS detection and
microscopic imaging capabilities.
In conclusion, MS is broadly and widely
used in drug discovery (for example in drug
target / biomarker discovery, protein / ligand
inter actions and quantification). The soft
ionisation techniques have opened a wide
opportunity to analyse macromolecules and our
ability to identify and quantify analytes has
greatly increased with the drastic improvement
in equipment design and capability and this is
highlighted by our ability to detect compounds
at the femtogram limit.
MASS SPECTROMETRY IN-DEPTH FOCUS
European Pharmaceutical Reviewwww.europeanpharmaceuticalreview.com Volume 17 | Issue 2 | 2012
07
Dr Min Yang attended the School ofChemistry and Chemical Engineering atNanjing University, graduating with aBSc in 1992 and an MSc in 1997, underthe supervision of Professor XiangzhenSun and Professor Yi Pan. He worked atthe Nanjing Chemical Plant from 1992
until 1994 and at Procter & Gamble Technology (Beijing) Co. Ltdfrom 1997 until 1999. From there he went to The School ofApplied Sciences, University of Huddersfield in Nov 1999 topursue the PhD under the supervision of Dr. Andrew Laws andProfessor Mike I. Page, being awarded his PhD in May 2003. Heundertook a postdoctoral position with Professor Benjamin G.Davis in the Chemistry Department, Oxford University from2002 until 2006. Then he moved to the Chemistry Departmentat Cambridge University in 2007 to undertake postdoctoralresearch project on microdroplets with Professor Chris Abell. In2007, he was appointed as a lecturer of mass spectrometry in the School of Pharmacy, which became UCL School ofPharmacy in January 2012.
BIOGRAPHY
Ana Rita Angelino undertook apharmaceutical sciences integratedMasters degree at the Faculty ofPharmacy, University of Lisbon,graduating as a pharmacist in 2012.During her degree, she was involved inseveral projects. In 2008/9, she
participated in the University of Lisbon/Amadeu DiasFoundation Investigation Grant with the project looking atmultidrug resistance in cancer under the supervision ofProfessor Maria José Umbelino Ferreira. In 2009/10, she wasinvolved in the Angelini University Award with the project‘miRNAs in Alzheimer’s Disease’, under the supervision ofProfessor Cecília Maria Pereira Rodrigues. She also developedtwo curricular projects related to the isolation andcharacterisation of bioactive natural products. In 2011, sheundertook a three-month ERASMUS placement at the School ofPharmacy, University of London, under the supervision of Dr.Min Yang. The project was related to a proteomic approach toidentify drug resistance targets in ovarian cancer cell lines.
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44. (a) Yang, M.; Proctor, M. R.; Bolam, D. N.; Errey, J. C.; Field,R. A.; Gilbert, H. J.; Davis, B. G. J. Am. Chem. Soc. 2005,127, 9336(b) Yang, M.; Davies, G. J.; Davis, B. G. Angew.Chem. Int. Ed. 2007, 46, 3885(c) Yang, M.; Brazier, M.;Edwards, R.; Davis, B. G. ChemBioChem 2005, 6, 346(d)Offen, W.; Martinez-Fleites, C.; Yang, M.; Lim, E.-K.; Davis,B. G.; Tarling, C. A.; Ford, C. M.; Bowles, D. J.; Davies, G. J.EMBO J. 2006, 25, 1396(e) Flint, J.; Taylor, E.; Yang, M.;Bolam, D. N.; Tailford, L. E.; Martinez-Fleites, C.; Dodson,E. J.; Davis, B. G.; Gilbert, H. J.; Davies, G. J. Nat. Struct.Mol. Biol. 2005, 12, 608(f) Bolam, D.; Roberts, S.; Proctor,M.; Turkenburg, J.; Dodson, E.; Martinez-Fleites, C.; Yang,M.; Davis, B. G.; Davies, G.; Gilbert, H. Proc. Nat. Acad. Sci.USA 2007, 13, 5336
45. (a) D'Alessandro, A.; Federica, G.; Palini, S.; Bulletti, C.;Zolla, L. Mol. Biosyst. 2011, online(b) Barr, J.; Vázquez-Chantada, M.; Alonso, C.; Pérez-Cormenzana, M.; Mayo,R.; Galán, A.; Caballería, J.; Martín-Duce, A.; Tran, A.;Wagner, C.; Luka, Z.; Lu, S.; Castro, A.; Le Marchand-Brustel, Y.; Martínez-Chantar, M. L.; Veyrie, N.; Clément,K.; Tordjman, J.; Gual, P.; Mato, J. M. J. Proteome. Res.2010, 9, 4501
46. (a) McDonnell, L. A.; Heeren, R. M. A. Mass Spectrom.Rev. 2007, 26, 606(b) Stoeckli, M.; Chaurand, P.;Hallahan, D. E.; Caprioli, R. M. Nat. Med. 2001, 7, 493
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Emerging from drug resistance observations in
cancer treatment, the relevance of drug
transporters for ADMET properties was
established first for the efflux transporter ABCB1
(alias MDR1 or P-gp). Today, a range of uptake
and efflux transporters are in focus for drug
development. The selection of clinically relevant
transporters listed in Table 1 (page 10) com -
prises representatives of efflux and uptake
transporters as prioritised by regulatory
authorities2,3. These transporters are found in
membranes of all pharmacokinetic (PK)-relevant
organs (intestine, liver, kidney, blood-brain
barrier (BBB)) as well as placenta, testis or lung
epithelia. Understanding the function of these
proteins as well as their expression levels in
tissues is critical to enable appropriate risk
assessments of transporter substrates during
drug development.
The relevance of the active transport
mechanism in the human clinical situation
needs to be assessed for all new medical entities
that are identified as drug transporter substrates
in vitro. While it is widely accepted that for highly
permeable and highly soluble drugs (class I
drugs according to the biopharmaceutics
classification system, BCS) the impact of active
transport on overall pharmacokinetics is
minimal, BCS class IV-drugs are dependent on
membrane transporters influencing all aspects
of their disposition4. Though understanding of
transporter functionality and advanced
substrate identification in vitro has continuously
progressed over the past few decades, there is
still a challenge to translate the in vitro findings
The importance of membrane bound drug transporters in drug absorption,distribution, metabolism, elimination, toxicity and efficacy (ADMET/efficacy) havebeen undoubtedly recognised by academia, the pharmaceutical industry andregulators1. With rapidly increasing knowledge on drug transporter functions in vitroand in vivo and advances in the field of bioanalytical methods, an opportunity toprogress in quantitative predictions of transporter interactions is evident. We willprovide the readers with an overview and discuss from a drug discovery perspectivethe benefit of being able to accurately measure the concentration of membranetransporters using liquid chromatography linked to tandem mass spectrometry (LC-MS/MS) with selected reaction monitoring (SRM).
QUANTIFICATION OF MEMBRANE DRUGTRANSPORTERS AND APPLICATION IN DRUG DISCOVERY AND DEVELOPMENT
Tasso Miliotis and Constanze HilgendorfInnovative Medicines, AstraZeneca R&D
European Pharmaceutical Reviewwww.europeanpharmaceuticalreview.com Volume 17 | Issue 2 | 2012
09
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miliotis SUPP_Layout 1 25/04/2012 14:04 Page 1
to the whole organ or to whole body PK.
Transporter substrates that are dependent on
multiple transporters in their disposition profile
are particularly challenging to quantitatively
predict from preclinical data.
What is the current interest inquantification?One key link that has been required to
extrapolate from the in vitro substrate param -
eters to the in vivo situation is appropriate
physiological scaling factors. There are well-
established procedures to extrapolate
metabolism from in vitro to in vivo with the help
of physiological scaling factors, e.g. microsomal
protein per gram tissue or hepatocellularity
(cells per gram liver), the tissue weight and
applying a well-stirred model for blood-flow to
the liver. In analogy, the first transporter scaling
methods were attempting to employ mg
transporter per gram tissue to scale up to organ
function. However, the nature of integral
membrane protein functioning as gatekeepers
in the lining membranes of organs implies that
determination of total protein amount per cell or
tissue preparation will rarely allow a relation to
functional membrane transporters.
Among in vitro systems to identify
transporter substrates as membrane vesicles,
cell lines or tissue specimens, only membrane
vesicle studies may acceptably express
transporter function in relation to total protein.
However, any cell-line or tissue based in vitro
system requires more complex characteri -
sations. In the case of tissue samples the cellular
surface exposed to the organ ‘outside’, e.g. to the
intestinal lumen or to the bloodstream
represents the relevant transporter containing
membrane. In a total protein determination
this membrane is virtually diluted with protein
from underlying cells or connective tissue which
are not contributing to the measured transport
activity and therefore do not appropriately
reflect the relevance of membrane transporters.
With respect to the in vivo human clinical
situation, transporter protein expression
variability among individuals can be an
important factor to understand variation in
pharmacokinetics. In addition to a good
knowledge of the qualitative setup of organ
transporter profiles, a continuous build of
quantitative transporter abundance in organs is
highly warranted5.
Quantitative protein determination in
membranes have predominantly used antibody-
based immunoassays, including Western
blotting of membrane preparations, fluor -
escence activated cell sorting (FACS)
methodologies, and enzyme-linked immuno -
sorbent assay (ELISA). A general challenge is the
preparation of specific antibodies, particularly if
the amino acid sequences of candidate proteins
are very similar. Though a large variety of
antibodies are commercially available, cross-
reactivity between protein isoforms or species
remains an issue. Furthermore, the development
of a high quality ELISA assay requires a significant
investment in time and resources and therefore
may not be readily accessible on a broad scale to
characterise in vitro systems and tissues in a
comparable manner. An additional complication
is that different preparation techniques may be
European Pharmaceutical ReviewVolume 17 | Issue 2 | 2012
10
MASS SPECTROMETRY IN-DEPTH FOCUS
Systematic name Common name Location, typical substrate chemotypes Clinical manifestationABCB1 P-gp Efflux of lipophilic compounds from brain endothelia, intestinal Limiting absorption and CNS exposure of substrates;
(P-glycoprotein, MDR1) enterocytes, hepatocytes (canalicular), kidney proximal tubule excretion in urine and bile. Role in multidrug resistance.
ABCG2 BCRP Efflux of compounds from intestinal enterocytes, brain endothelia, Limiting absorption and CNS exposure of substrates; excretion in bile. mammary glands, placenta, hepatocytes (canalicular), stem cells Role in multidrug resistance. Clinically relevant polymorphisms.
ABCB11* BSEP Efflux from hepatocytes (canalicular), toxicologically Role in excretion of bile acids, inhibition may cause cholestasis.relevant transporter Clinically relevant polymorphisms.
SLCO1B1 OATP1B1 Uptake of anionic compounds across sinusoidal liver membrane. Role in hepatic disposition and excretion.Substrate overlap with OATP1B3 Clinically relevant polymorphisms.
Transport of bile acids/bilirubin.SLCO1B3 OATP1B3 Uptake of anionic compounds across sinusoidal liver membrane. Role in hepatic disposition and excretion. Transport of bile acids.
Substrate overlap with OATP1B1SLC22A1* OCT1 Uptake of small cationic compounds across sinusoidal liver Role in hepatic disposition and excretion.
membrane, intestinal enterocytesSLC22A2 OCT2 Uptake of small cationic compounds into kidney proximal tubule, neurons Role in renal excretion. Role in creatinine uptake. SLC22A6 OAT1 Uptake of small anionic compounds into kidney proximal tubule, placenta Role in renal excretion. SLC22A9 OAT3 Uptake of small anionic compounds into kidney proximal tubule, Role in renal excretion.
choroid plexus, BBB
TABLE 1 The key transporters of interest to drug disposition as prioritised by the EMA and FDA, modified from 1; * transporter prioritised by EMA only.
FIGURE 1SRM analysis on a triple quadrupole tandem mass spectrometer. The mass spectrometer is set up to perform a SRM analysis in which specific precursor(s)-to-product ion transition(s) is measured. In Q1 mass filter the targeted analyte(s) is selected on the basis of its mass over charge ratio (m/z). Q2 fragments the selectedanalyte(s) and in Q3, the optimised fragments are filtered out and finally detected. The end result is a highly selective mass spectrum recorded on a chromatographictime scale
miliotis SUPP_Layout 1 25/04/2012 14:04 Page 2
required for different sample types, or when a
comparison between different species is
needed. The different affinities of specific
antibodies against transporters in different
species require specific calibrations using
purified proteins that are often lacking. Thus,
immunobased methods are less versatile in
application to cross-system scaling.
Mass-spectrometric quantification of transporters The recent technology advancements in mass
spectrometry instrumentation have enabled the
development of highly specific and sensitive
assays for quantification of many proteins in a
single measurement6. The use of LC-MS/MS has
emerged as viable alternative to classical
methods of protein quantifications and
facilitated the development of protein
quantification assays in a time- and cost-
effective manner. The absolute quantification of
proteins in biological samples is based on the
well-established isotope dilution concept7.
Defined quantities of isotope-labelled standards
that exhibit chromatographic behaviours
identical to the native target analyte(s) are
added to the sample, and the label permits that
they are easily distinguished by the mass
spectrometer through their mass difference.
Traditionally, quantification by LC-MS/MS
for small molecular compounds has been
accomplished using triple quadrupole (QQQ)
instruments operated in SRM mode. In contrast
to small molecules, the detection sensitivity of
large proteins is limited by the size of the
molecule and the wide distribution of protein
charge states. Moreover, the range of the mass
filter on typical QQQ instruments is restricted to
a mass-to-charge ratio (m/z) of about 1500-
2000. Hence, it is essential to digest the protein
in a first step with a protease, such as trypsin,
and identify appropriate signature peptide(s) for
each target protein(s). These peptides have been
termed as proteotypic peptides and they are
characterised by their sequence uniqueness in
the context of a particular proteome and their
efficient mass spectrometric detection8.
The LC-SRM analysis is schematically
illustrated in Figure 1 opposite. In SRM mode,
the first and third quadrupoles act as mass filters
to specifically select predefined m/z values of
the proteotypic peptide(s) and its corre -
sponding specific fragment ion(s). In the second
quadrupole, the target peptide(s) is fragmented
by collision excitation with a neutral gas. The use
of two mass filters provides a high signal-to-
noise ratio and high selectivity. Modern QQQ
mass spectrometers with fast spectral scan rate
allow large numbers of SRM acquisitions in a
duty cycle and numerous data points across
a chromatographic peak for accurate,
precise quantitation of many proteins within a
single LC-MS/MS run without compromising the
detection sensitivity. In the ADMET field it has
demonstrated the simultaneous quantifica-
tion of 37 membrane bound proteins (e.g.
transporters, ion-channels)9.
The general description of protein
quantification using a mass spectrometric
strategy is outlined in Figure 2. Excellent reviews
regarding the details of establishing an SRM
experiment for protein quantification have
been published10,11. Briefly, the critical step in
the experimental design is the selection of the
proteotypic peptides that will act as surrogate
markers for the target protein(s) since it will
affect the specificity as well as the sensitivity of
the LC-SRM assay. The addition of a stable
isotope-labelled peptide, typically 13C and 15N, is
introduced as an internal standard as early as
possible into the sample at a known and fixed
concentration. The quantification biases are
minimised since this internal peptide standard
shares the same physicochemical properties as
the endogenous targeted peptide, including
chromatographic co-elution, ionisation efficiency
and fragmentation pattern. The following factors
should be addressed when considering an
appropriate proteotypic peptide candidate: the
uniqueness of a peptide to the corresponding
target protein, the physico chemical properties
of a peptide including hydro phobicity and
ionisation efficiency, known post translational
modifications and known amino acid variants.
Additionally, peptides with reactive or labile
amino acid residues should be avoided,
particularly methionine, cysteine and tryptophan
are prone to oxidise while Asp-Pro and Asp-Gly
bonds are unstable.
Finally, monitoring the efficiency and
reproducibility of the tryptic digestion of the
target protein(s) is essential for ensuring
accuracy in the LC-SRM methodology. However,
it is seldom the case that purified membrane
transporters are commercially available
MASS SPECTROMETRY IN-DEPTH FOCUS
European Pharmaceutical Reviewwww.europeanpharmaceuticalreview.com Volume 17 | Issue 2 | 2012
11
FIGURE 2 General workflow of LC-SRM based quantitative strategy. The procedure consists essentially oftwo stages. The first stage involves the selection of a proteotypic peptide(s) that act as a surrogatemarker(s) for the target protein(s), the generation of stable isotope labelled peptides and the subsequentoptimisation of the LC-SRM analysis. Finally, in the second stage the developed LC-SRM protocol is applied to the sample containing the target protein(s). Note that a protease digestion is an essential part ofthe sample preparation. A stable isotope labelled peptide having the same amino acid sequence as the proteotypic peptide is added as internal standard at a fixed concentration as early as possible. Theabsolute quantification is determined by plotting the ratio of the peak areas of the proteotypic peptide tothe internal standard (y) against the concentration of a synthetic endogenous peptide (x) for constructingthe regression analysis
miliotis SUPP_Layout 1 25/04/2012 14:04 Page 3
for calibration and alternative validation
procedures to monitor digestion efficiency have
been developed. Li et al included a surrogate
digestion substrate peptide that comprised the
amino acid sequence of the proteotypic
peptide, which was flanked with tryptic
cleavage sites and additional amino acid
residues12. Another reproducibility aspect that
needs to be addressed is related to membrane
preparation itself and intra- and inter-sample
variability, which increases between different
sources of tissue. Terasaki and co-workers have
shown that Na-K ATPase is stably expressed in
the plasma membrane of different cell types and
species and therefore may be successfully
applied to normalise between different
membrane preparations13.
Application of membrane transporterquantification in ADMET The application of quantitative multiplexed
LC-SRM analysis of membrane proteins in
ADMET related studies has increased
significantly during the last few years. Several
publications described the development of
multiplexed LC-SRM assays for quantification of
membrane proteins and by using this
methodology they constructed a quantitative
atlas of membrane transporter proteins at the
blood-brain barrier, liver and kidney in mice9,14.
Niessen et al combined RT-PCR measurements
and subsequent LC-SRM analysis of ABC
transporters as an effective strategy to identify
and quantify membrane proteins expressed in
target cells and tissues, further intracellular
localisation was confirmed with immuno -
histochemistry15. Application of LC-SRM
to transporter quantification and the use of
protein amount in functional analysis are
demonstrated in recent works by Miliotis et al
and Li et al16,17. Miliotis et al developed an LC-SRM
assay for the ABC transporter P-gp (MDR1) and
quantified the amounts in Caco-2 cell
monolayers and in inside-out membrane
vesicles16. The results showed a good correla-
tion between the function and the P-gp
content in the tested in vitro systems and
confirmed that the P-gp concentration is directly
related to the level of activity in the respective
system. In the study by Li et al, the application of
absolute transporter quantification data
in sandwich cultures could support the
quantitative evaluation of different biliary
cleared compounds17.
Additionally, a significantly improved
understanding of species differences between
humans and animals can be attributed to LC-
SRM measurements of membrane transporters.
Terasaki and co-workers have measured the
absolute protein expression levels of
transporters in human, monkey, and mouse
brain capillaries18,19. The results gave indications
that average BCRP and P-gp transporter
expression at the human BBB are similar. In mice,
the P-gp analogue abcba1 was more than
threefold higher expressed than mouse bcrp19.
These inter-species comparisons have shown
that in addition to differences in substrate
specificity and affinity large differences in
expression levels also affect variability between
species with regard to transport at the BBB.
Taking into account the observed species
differences in protein abundance will improve
our ability to cross-species translate exposure in
the central nervous system from mice to man.
European Pharmaceutical ReviewVolume 17 | Issue 2 | 2012
12
MASS SPECTROMETRY IN-DEPTH FOCUS
1. Giacomini, K.M., Huang, S., Tweedie, D.J., Benet, L.Z.,Brouwer, K.L.R., Chu, X., Dahlin, A., Evers, R., Fischer, V.,Hillgren, K.M., Hoffmaster, K.A., Ishikawa, T., Keppler, D.,Kim, R.B., Lee, C.A., Niemi, M., Polli, J.W., Sugiyama, Y.,Swaan, P.W., Ware, J.A., Wright, S.H., Yee, S.W., Zamek-Gliszczynski, M.J., Zhang, L., The InternationalTransporter Consortium, Membrane transporters indrug development, Nature Reviews Drug Discovery 9,215-236, 2010
2. European Medicines Agency (EMA), Guideline on the Investigation of Drug Interactions,CPMP/EWP/560/95/Rev. 1, April 2010:(available athttp://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2010/05/WC500090112.pdf)
3. US Food and Drug Administration (FDA). (Draft 2012)Guidance for Industry Drug Interaction Studies —Study Design, Data Analysis, Implications for Dosing,and Labeling Recommendations :(available athttp://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM292362.pdf)
4. Wu, C.Y., Benet, L.Z., Predicting drug disposition viaapplication of BCS: transport/absorption/ eliminationinterplay and development of a biopharmaceuticsdrug disposition classification system, PharmaceuticalResearch 22, 11-23, 2005
5. Nilsson P., Paavilainen, L., Larsson, K., Ödling, J.,Sundberg, M., Andersson, A., Kampf, C., Persson, A., Al-Khalili, S., Ottosson, J., Björling, E., Hober, S., Wernérus,H., Wester, K., Pontén, F., Uhlen, M., Towards a humanproteome atlas: High-throughput generation of mono-specific antibodies for tissue profiling, Proteomics 5,4327-4337, 2005
6. Whiteaker, J.R., Lin, C., Kennedy, J., Hou, L., Trute, M.,Sokal, I., Yan, P., Schoenherr, R.M., Zhao, L., Voytovich,U.J., Kelly-Spratt, K.S., Krasnoselsky, A., Gafken, P.R.,Hogan, J.M., Jones, L.A., Wang, P., Amon, L., Chodosh,L.A., Nelson, P.S., McIntosh, M.W., Kemp, C.J.,Paulovich,A.G., A targeted proteomics–based pipeline forverification of biomarkers in plasma, NatureBiotechnology 29, 625-634, 2011
7. Bowers, G.N., Fassett, J.D., White, E., Isotope dilutionmass spectrometry and the National Reference System,Analytical Chemistry, 65, 475R-479R, 1993
8. Mallick, P., Schirle, M., Chen, S.S., Flory, M.R., Lee, H.,Martin, D., Ranish, J., Raught, B., Schmitt, R., Werner, T.,Kuster, B., Aebersold, R., Computational prediction ofproteotypic peptides for quantitative proteomics,Nature Biotechnology, 25, 125-131, 2007
9. Kamiie, J., Ohtsuki, S., Iwase, R., Ohmine, K., Katsukura,Y., Yanai, K., Sekine, Y., Uchida, Y., Ito, S, Terasaki, T.,Quantitative atlas of membrane transporter proteins:development and application of a highly sensitivesimultaneous LC/MS/MS method combined with novelin-silico peptide selection criteria, PharmaceuticalResearch, 25, 1469-1483, 2008
10. Lange, V., Picotti, P., Domon, B., Aebersold, R., Selectedreaction monitoring for quantitative proteomics: atutorial, Molecular systems Biology, 4, 1-14, 2008
11. Pan, S., Aebersold, R., Chen, R., Rush, J., Goodlett, D.R.,McIntosh, M.W., Zhang, J., Brentnall, T.A., Massspectrometry based targeted quantification: methodsand applications, Journal of Proteome Research, 8, 787-797, 2009
12. Li, N., Nemirovskiy O.V., Zhang, Y., Yuan, H., Mo, J., Ji, C.,Zhang, B., Brayman, T.G., Lepsy, C., Heath, T.G., Lai, Y.,Absolute quantification of multidrug resistance-associated protein 2 (MRP2/ABCC2) using liquidchromatography tandem mass spectrometry,Analytical Biochemistry, 380, 211-222, 2008
13. Ohtsuki, S., Schaefer, O., Kawakami, H., Inoue, T., Liehner,S., Saito, A., Ishiguro, N., Kishimoto, W., Ludwig-Schwellinger, E., Ebner, T., Terasaki, T., SimultaneousAbsolute Protein Quantification of Transporters,Cytochromes P450, and UDP-Glucuronosyltransferasesas a Novel Approach for the Characterization ofIndividual Human Liver: Comparison with mRNA Levelsand Activities, Drug Metabolism and Disposition 40, 83-92, 2012
14. Ohtsuki, S., Uchida, Y., Kubo, Y., Terasaki, T., Quantitativetargeted absolute proteomics-based ADME research
as a new path to drug discovery and development:methodology, advantages, strategy, and prospects.Journal of Pharmaceutical Sciences, 100, 3547-3559, 2011
15. Niessen, J., Jedlitschky, G., Grube, M., Kawakami, H.,Kamiie, J., Ohtsuki, S., Schwertz, H., Bien, S., Starke, K.,Ritter, C., Strobel, U., Greinacher, A., Terasaki, T., Kroemer,H.K., Expression of ABC-type transport proteins inhuman platelets, Pharmacogenetics and Genomics 20,396-400, 2010
16. Miliotis, T., Ali, L., Palm, J.E., Lundqvist A.J., Ahnoff, M.,Andersson, T.B., Hilgendorf, C., Drug Metabolism andDisposition, 39, 2440-2449, 2011
17. Li, N., Singh, P., Mandrell, K.M., Lai, Y., Improvedextrapolation of hepatobiliary clearance from in vitrosandwich cultured rat hepatocytes through absolutequantification of hepatobiliary transporters. MolecularPharmacology 7, 630-641, 2010
18. Ito, K., Uchida, Y., Ohtsuki, S., Aizawa, S., Kawakami, H.,Katsukura, Y., Kamiie, J., Terasaki, T., Quantitativemembrane protein expression at the blood-brainbarrier of adult and younger cynomolgus monkeys,Journal of Pharmaceutical Science, 100, 3939-3950, 2011
19. Uchida, Y., Ohtsuki, S., Katsukura, Y., Ikeda, C., Suzuki, T.,Kamiie, J., Terasaki, T., Quantitative targeted absoluteproteomics of human blood-brain barrier transportersand receptors, Journal of Neurochemistry, 117, 333-345, 2011
20. Li, N., Zhang, Y., Hua, F., Lai, Y., Absolute difference ofhepatobiliary transporter multidrug resistance-associated protein (MRP2/Mrp2) in liver tissues andisolated hepatocytes from rat, dog, monkey, andhuman, Drug Metabolism and disposition, 37, 66-73, 2009
21. Li, M., Yuan, H., Li, N., Song, G., Zheng, Y., Baratta, M.,Hua, F., Thurston, A., Wang, J., Lai, Y., European Journal ofPharmaceutical Sciences, 35, 114-126, 2008
REFERENCES
miliotis SUPP_Layout 1 25/04/2012 14:04 Page 4
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Similar advances in the quantification of hepatic
efflux transporters have been reported by Li et al
who measured the concentration of MRP2 in
liver tissues and isolated hepatocytes across rat,
dog, monkey and human20. They showed that
MRP2 protein expression in rat liver was about
tenfold higher as compared to the other species.
Li et al investigated the interspecies difference in
efflux transporter activity in hepatocytes from
dog, rat, monkey and human and observed
highest transport activity of specific MRP2
substrates in rat hepatocytes, fourfold higher
compared to human21. Rat, monkey and human:
MRP2 activity ranked in accordance to the
protein abundance measurements. In contrast,
dog hepatocytes demonstrated MRP2 mediated
efflux similar to rat hepatocytes, despite lower
protein levels. This underlines that protein
abundance needs to be used in combination
with substrate specificity (Km) data to scale the
overall functional differences of MRP2 efflux
between the investigated species.
Conclusion The fundamental scientific progress in whole
genome sequencing and bioinformatics
together with the introduction of the latest
generation of mass spectrometers, have
facilitated the development of highly specific
and sensitive MS-quantifications of membrane
transporters. LC-MS/MS methodologies offer
time- and cost-effective protein quantifications
and have emerged as a viable alternative to
immunobased methods. This technology
development is seemingly closing a gap in the
capability to enable cross-species scaling and
in vitro to in vivo extrapolation of the quanti -
tative relevance of drug transporters to
disposition / ADMET. While essential knowledge
on the transporter substrate status is readily
available, the relative contributions of active (via
transporters) and passive distribution processes
are more challenging to estimate. Incorporating
absolute transporter abundance in different in
vitro systems and PK-relevant organs is a
requirement for successful translations from
in vitro to in vivo. Here, the application of novel
quanti tative methods such as mass spectro -
metry provides an important contribution to
advance our ability to quantitatively extrapolate
transporter function to the clinical situation
in the future.
MASS SPECTROMETRY IN-DEPTH FOCUS
Dr. Tasso Miliotis completed his PhD in analytical chemistry at LundUniversity in Sweden in 2001. He thendid postdoctoral studies at AstraZenecaR&D Mölndal where he developed amass spectrometric based platform thatwas used for de-orphanisation of GPCRs.
Tasso has remained at AstraZeneca R&D Mölndal and workstoday as an Associate Principle Scientist in Translational ScienceCVGI iMED. He focuses on assay development and innovativeresearch for biomarker discovery.
BIOGRAPHY
Dr. Constanze Hilgendorf is currentlya Principal Scientist Drug Disposition inthe Global DMPK Centre of Excellence,Innovative Medicines, AstraZeneca R&DMölndal. Constanze received a PhD fromthe faculty of Pharmaceutical Chemistryand Pharmacokinetics at University
Halle-Wittenberg, Germany with research on cell-basedintestinal permeation and transport models. After working incontract research in Germany, she joined DMPK at AstraZenecaMölndal, Sweden in 2002. She held various project andscientific roles, yielding broad experience across DMPKdisciplines in early and late phases of drug development. Her core scientific interests are translatable in vitro ADMEmodels, drug transporters, interindividual variability and DDI predictions.
BIOGRAPHY
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European Pharmaceutical ReviewVolume 17 | Issue 2 | 2012
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More than 6,500 scientists are expected to
attend the conference this year, and nearly 3,000
papers will be presented over the five days,
either as part of the short courses – which
precede the event, starting on 19 May – or as
poster presentations in and around the
convention centre.
The five-day programme will begin on
Sunday 20 May with tutorials from Jentaie Shiea,
National Sun Yat-Sen University and Keith A.
Baggerly, MD Anderson Cancer Centre, who will
present on ‘Ambient Mass Spectrometry:
Analysis in the Real World by a ‘Green’
Technology’ and ‘Statistics and Forensic
Bioinformatics: Analytic Issues in High-
Throughput Biology’, respectively. The tutorials
will be followed by an opening session and a
plenary lecture led by Chris Reddy from
Woods Hole Oceanographic Institution,
who will present a lecture entitled ‘The
Deepwater Horizon Oil Spill: From the Pipe to
the Plume’. A welcome reception will bring a
close to the first day.
The following four days will consist of full
programmes of concurrent oral sessions. Almost
50 oral sessions – some of which co-chaired by
leading organisations in the mass spectrometry
field across the globe, including the Australian,
New Zealand, Japanese, Korean, and Hong Kong
MS Societies – will take place over the four days
covering a range of topics. The sessions include:
l Disease Biomarkers and Pathways
l Biomarkers in Drug Discovery
and Development
l Time-of-Flight Mass Spectrometry:
New Developments in Instrumentation
and Applications
l Radical-driven Peptide Fragmentation
l Post-translational Modifications:
Beyond Phosphorylation
l Quantification of Targeted Proteins and
Post-translational Modifications
l Advances in Nano-scale Separations for
MS Analysis
l Metabolomics: Clinical Applications
l Drug and Metabolite Analysis: Novel
Approaches for Dried Biological Samples
l Fundamentals of Peptide Fragmentation
l FAIMS and DMS: New Developments
and Applications
l PTMs: Comprehensive Analysis and
Combinatorial Patterns
l Environmental Contaminants: The Role of
MS in the 21st Century
To ensure visitors and delegates get the most
out of the event, workshops and interest
group meetings will be held each day after the
oral sessions.
As an added bonus for attendees of this
year’s event, some ASMS exhibitors will be
holding breakfast seminars during the
conference week; these include IonSense
Inc, Biotage, Tosoh Bioscience and Agilent
Technologies. Visitors will need to pre-register to
attend these, which can be done via the website:
www.asms.org/conferences/annualconference.
Poster presentations will also play an
important role in the 60th Annual Conference
on Mass Spectrometry and Allied Topics.
An effective way to communicate research to
colleagues, over recent years it has become the
‘presentation of choice’ for many scientists
exhibiting at ASMS and the Annual
Conference is renowned for the quality of the
posters presented.
The Conference will conclude on Thursday
24 May with a plenary lecture from well-known
food scientist, TV personality and author, Shirley
O. Corriher. Entitled ‘The Secret Life of Food’, the
lecture promises to be both interesting and
entertaining. This will be followed by a Closing
Gala at the Convention Centre, for which a
ticket is required.
The Annual Conference on Mass Spectro -
metry and Allied Topics, as always, looks set to be
an unmissable industry event. The five days will
provide the perfect backdrop for those looking
to network and learn from industry peers –
many of whom are leading research and
development scientists.
The 60th Annual Conference on Mass Spectrometry and Allied Topics will be takingplace between 20 – 24 May 2012 in Vancouver, Canada. Sponsored by the AmericanSociety for Mass Spectrometry, the event is one of the most dynamic scientificconferences in the world; aiming to promote and disseminate knowledge of mass spectrometry.
SHOW
PREVIEWDate 20 – 24 May 2012VenueVancouver Convention Centre, Vancouver, Canada
To find out more information about getting to the event, view the full programme
or to register your attendance, view the website:
www.asms.org/conferences/annualconference
FURTHER INFORMATION
asms SUPP_Layout 1 25/04/2012 14:05 Page 1
Innovation in MSinstrumentation continues togo from strength to strength(Orbitrap, multipass TOF, IMS-MS, miniaturisation).Where do you see the nextopportunities for innovation?
Baldi: There is increased demand in the market
for alternative sample introduction tech -
nologies. This is driven by high throughput
applications as well as the need to analyse the
distribution profile of molecules in a sample.
Direct ionisation of molecules from tissues or
liquid samples can modify and streamline
laboratory workflows and provide a unique level
of information. We see a lot of focus in this area
from different academic groups as well as bio-
tech companies.
Harland: Innovation can come in various guises.
We are now at a point where we can transfer
nearly all of the captured ions through the mass
spectrometer with very fine control, and we
continue to find ways to better deliver
sensitivity, selectivity and speed. A more
efficient generation of ions in the first place is
obviously an area of great opportunity, but also
represents a technology foundation that will
spur further advancement. I think we will see
innovations in delivering this performance in
smaller instruments, and in systems that are
more fully integrated for their specific end-use.
Hochmuth: We perceive a significant mis -
balance between hardware and software
advancements. Hardware development shows
very fancy and innovative solutions, but most
mass spec software is still general-purpose style,
no matter how specific the requirements of a
certain user are. Thus, our focus is on how
analytical data are interpreted, quantitated and
documented. Custom-made software offers
incredible improvements in terms of reliability,
efficiency and quality. Innovation in software is a
whole new dimension still widely unexplored
and with an excellent return of investment.
McLeod: There is great excitement about
MS as an imaging technique. The ability to
relate MS information directly to biology
and tissue structure has important applica-
tions in understanding drug distribution
and metabolism, as well as histology and
proteomics. For MS/MS, ETD (electron transfer
dissociation) has already opened up the study of
modifications in proteins; we expect novel
fragmentation techniques to be in demand.
European Pharmaceutical Reviewwww.europeanpharmaceuticalreview.com Volume 17 | Issue 2 | 2012
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LEADERS ROUNDTABLE
Moderator:Anthony Bristow, AstraZeneca
George McLeodMarket Manager, Pharmaceutical MS,
Bruker Daltonics
Detlev HochmuthScientific Consultant and Software
Developer for MS andChemoinformatics, Dr. Hochmuth
Scientific Consulting
Alessandro BaldiVP & General Manager, Protea Biosciences Inc
Gary HarlandMass Spectrometry Product Manager,
Waters Corporation
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A third interesting concept is the drive to
ultra-high resolution MS, challenging the need
for high-resolution separations and accessing
whole new levels of detail in large biomolecules.
Separation science continues to strive for faster separationswith even greater efficiencyand resolution. How will MSdevelopment meet thedemands of these separations?
McLeod: Here, the key requirement is to obtain
all the necessary information from a fast-
changing signal – whether introduced by LC, GC
or even CE. The goal then is to operate at speed
whilst retaining vital capabilities such as mass
accuracy, high dynamic range and fast MS/MS.
The stability of the signal under fast analysis is
also critical – resolution and accuracy, plus
information such as the isotopic pattern, must
be robust. This is what the next generation of
successful instruments will deliver.
Harland: MS acquisition speed has undoubtedly
been set a challenge in recent years by new
separations tools, such as UPLC. As a systems
manufacturer we are in a fortunate position to
be able to align our development efforts
through the complete analytical system to
ensure a new capability is realised by all
components. Not only the separation speed, but
the requirement to ask more questions of each
peak also demands that MS is ahead in its ability
to perform multiple experiments in parallel.
Baldi: We feel that the major area of innovation
to address fast separations would be in the
development of array-based detectors. Since
mass spectrometers are used more and more for
specific applications, an array-based detector
would address both high throughput LC/MS
applications as well as demanding sample
mapping analysis.
Hochmuth: In my view, mass spectrometric
acquisition speed is currently able to cope
with the chromatographic developments.
Ease of use and excellent long-term reliability of
fast separation techniques is the greater
challenge. Software innovation like optimised
and flexible acquisition methods with much
easier setup of complex scan conditions will be a
key factor to adapt to rapid analyses. Hardware
manufacturers need to evolve established
acquisition solutions to meet up with modern
demands of flexibility and speed.
In LC-MS, the Holy Grail is atruly universal ionisationsource. Are we getting closer tothe realisation of this dream?
Baldi: The demand of universal sources goes
hand-in-hand with the continuous demand for
additional sensitivity. These two drivers are
unfortunately going in the opposite direction.
Although we can advocate for a universal
source, there will always be specific applica-
tions / molecules demanding for specific
ionisation technologies.
Hochmuth: Honestly, I do not see a ‘truly
universal’ ionisation source in the near future.
Sure, there are noteworthy advancements, but
considering various matrix effects and complex
mixtures, I wonder whether more selective
ionisation methods wouldn't be the better
direction to go, at least as long as those methods
are studied and well understood. We do not
need to ionise whatever is there; in contrast, we
should try to view non-universal ionisation as
part of the separation and enrichment process,
i.e. to come to terms with it as friend not foe.
Insight into and knowledge of details of the
ionisation processes are key factors.
Harland: Closer? Yes. How close, is the question.
We are certainly on the road to being able to
handle much greater chemical diversity from a
single ionisation source, but research takes time
to become a commercial reality (and become
accepted by the community). Waters’ modular
open architecture source design however does
allow multiple ionisation modes to be usable
within minutes, from direct analysis to coupling
LC, SFC or GC, and is specifically designed to
allow new, emerging techniques to be quickly
adapted to our instruments for evaluation.
McLeod: In short: no! If anything, we have more
choices than ever. Developments such as heated
electrospray and CaptiveSpray have extended
the flow range, robustness and ionisation
capability of the workhorse LC-MS sources.
Beyond those, there are major innovations using
MALDI for drug distribution imaging,
European Pharmaceutical ReviewVolume 17 | Issue 2 | 2012
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MASS SPECTROMETRY IN-DEPTH FOCUS LEADERS ROUNDTABLE
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microbiology and even high-through put
screening. EI and CI remain vital techniques.
Niche sources such as cryospray, APLI and APPI,
and techniques for none-solution phase work
will likely proliferate. Finally, don’t overlook the
very different needs of inorganic chemists. There
is no ‘Swiss army knife’ source that’s better than
the proper tool for the job.
MS instrumentation continuesto become more robust anduser friendly and thereforemore readily available to thenon-expert, resulting in a much broader user base.Therefore, how do we develop these new users inarguably the more challengingareas of data interpretation,method development andtrace level quantitation?
Hochmuth: This is the key field of our company:
optimised, custom-made and easy to use
applications with user interfaces worth the
name, i.e. made for the user, not for the system.
In an age of modern applications like tablet and
smartphone apps, it is a shame that desktop
software often still relies on non-intuitive,
overloaded, complicated interfaces. Smart,
tailor-made software with workflow and layout
adapted to specific customers, purposes and
laboratory workflow is the solution we offer,
performed by mass spec experts rather than
general-purpose programmers.
Harland: You could argue a case for greater MS
education of users, but ultimately solely
focusing on technology education is not going
to be appropriate for many of the emerging
applications of mass spectrometry. Just like the
developments to make the instruments easier to
use, the method development and data
interpretation has to be part of what the system
provides to the user. If we are to take things to
their logical course, MS-based systems need
to provide the answer to non-experts, not rely
on their skill to interpret. Of course, that's not to
say that a true expert will not be able to realise
better results if they spend the time, but we
need to aim for non-experts all getting the
same consistent answer out of a system.
McLeod: The broadening user base is a trend we
as manufacturers must embrace. Do all of these
users want or need development in the more
esoteric aspects of mass spectrometry? They
may be scientists with their own demanding
specialism to concentrate on. They may be
analysts with deadlines to meet. More likely,
they want a tool, a service or an information
machine. To satisfy these users, we must develop
the experience of handling MS instruments and
data. Both software and automation will evolve
to be more intelligent and increasingly
applications-led.
Baldi: Sample preparation / introduction is still
one of the major barriers and source of errors
for non-expert users. We, as suppliers, are
continuously working to facilitate user
interaction with the instruments, sometimes
mimicking well established / known workflows.
For instance, in our direct ionisation system the
LAESI DP-1000, we are facilitating MS-based
mapping providing a software workflow which
replicates that of optical microscopes for
selection of an area within a tissue or cell
population for molecular and distribution
profiling. This provides a better access to
customers that are not coming from the
analytical chemistry world but are still in need of
mass spectrometry-based data.
The applications of MScontinue to become broaderand even more inspirational(for example, real time MSmonitoring in the operatingtheatre and as a clinicaldiagnostic tool). Where nextcan MS have an impact?
McLeod: The clinical aspect you mention is
pivotal. Point-of-care clinical analysis has the
potential to both improve patient care and
deliver long-term cost savings. It ties in very
much with earlier questions – MS in this field
must be recognised as a trusted, intelligent
partner. We are already seeing it happen with
microbiology and there is huge potential in
fields such as histology and toxicology. Mass
spectrometry can become truly inspirational
when we see it saving lives.
Baldi: Mass spectrometry is indeed becoming a
required tool for many clinical diagnostic
applications. When combined with specific
sample introduction / sample mapping
technologies, mass spectrometry can be
applicable in surgical pathology and more
general histo-pathology. It has the ability to
provide objective molecular data versus
subjective optical information, which is critical
to enhance the quality of the information.
Hochmuth: Right, currently, life science and
clinical applications are clearly the strongest
market of mass spectrometry evolution. We also
see miniaturisation and in-field use by non-
experts as an important area which will have
great impact of how mass spectrometry will be
used in the near future for a wide variety of
monitoring and analytical tasks. Some very
interesting developments are going on even
right now.
Harland: The ideas are endless; it's a case of
when the technology and the application can
come together. Can we put the performance
needed into the hands of that user and the
required environment at the right cost?
Any point of care or in situ sampling situation,
such as the clinic, is an area where MS can have a
real impact.
European Pharmaceutical Reviewwww.europeanpharmaceuticalreview.com Volume 17 | Issue 2 | 2012
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