MASS SPECTROMETRY - European Pharmaceutical Review · European Pharmaceutical Review ... types of mass analysers. Tandem Mass Spectrometry (MS/MS) ... added isotope labelling by amino

Proteomics, small molecules and metablomics 03

Membrane drug transporter quantification 09

The 60th annual conference on Mass Spectrometry

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Anthony Bristow, AstraZeneca, poses the questions for our

Mass Spectrometry Leaders Roundtable 15

IN-DEPTH FOCUS

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European Pharmaceutical Reviewwww.europeanpharmaceuticalreview.com Volume 17 | Issue 2 | 2012

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MASS SPECTROMETRY

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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


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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

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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 ”


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


06


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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 ”


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.



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.

[email protected]

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


bruker_Layout 1 10/04/2012 09:56 Page 1

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


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


10


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


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



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


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.


12


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


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Terpenoids libraryMS library incl. retention indices for essential oil and flavour analysis

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.


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



14

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.


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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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mass spec roundtable SUPP_Layout 1 25/04/2012 14:06 Page 1

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,


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MASS SPECTROMETRY IN-DEPTH FOCUS LEADERS ROUNDTABLE


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.


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