Westermeier R., Naven T., H?pker H.-R. Proteomics in Practice: A Guide to Successful Experimental Design

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3 Mass Spectrometry220

Matrix Matrix Structure Application

2,4,6-Trihydroxyaceto-

phenone (THAP)

UV laser: oligo-

nucleotides <3 kDa

3-Hydroxy picolinic

acid

COOH

UV laser: Oligo-

nucleotides >3 kDa

Due to the pulsed nature of MALDI, it has been predominantly

coupled with the pulsed, time-of-flight (TOF) analyzer or configura-

tions thereof. This combination, with its simple operation, ease of

automation and robust performance has meant that MALDI-TOF

instruments are common place for routine protein identification

such as in the 2D PAGE-MS workflow where protein spots are picked

from a 2DE gel (see Section 1.7.1). The use of MALDI-TOF for pep-

tide mass fingerprinting (PMF) is more than sufficient for protein

identification from these simple samples.

However, MALDI PMF is not a suitable method for protein identifi-

cation when a complex mixture is under investigation. For the analyis

of more complex samples, tandem mass spectrometry (MS/MS) is

almost exclusively required and there is an absolute need to couple a

chromatographic step with MS. MALDI has long been incompatible

with liquid chromatography and as such a chromatographic step has

typically been coupled with electrospray ionization equipped mass

spectrometers for MS/MS applications. However, the innate features

of MALDI still make it a very attractive and accessible technique.

Thus, the introduction of the MALDI TOF/TOF configuration with

its high performance MS/MS specifications and recent developments

in liquid handling systems enabling nano-scale chromatography pep-

tide separations to be coupled with MALDI system capable of

MS/MS, have been a welcome addition to the proteomics toolbox.

Importantly, this configuration supports both high performance PMF

and MS/MS in a single system.

In this offline method, LC-MALDI, peptides are eluted from a

nano-LC column in the standard fashion, but are then mixed with a

matrix prior to spotting onto a MALDI plate. In a standard configura-

Pieles U, Zurchner W, Schar

M, Moser HE. Nucleic Acids

Res 21 (1993) 3191–3196.

3.1 Ionization 221

tion, such an LC-MALDI spotter will consist of a T-piece, syringe

pump, and micro fan. A low dead-volume PEEK-T and mixer (dead

volume <50 nL) connect the capillaries from the syringe pump and

UV cell to the sample needle, providing reproducible mixing before

spotting. The syringe pump delivers the matrix solution with contin-

uous flow rates down to a few nanoliters per minute. Different syr-

inge barrel sizes can be employed, eliminating the need for frequent

refilling of small syringes. The length of the capillary connections

from the UV cell and the syringe pump are short, resulting in a delay

time as short as 3 minutes. Commercial LC-MALDI spotters are pro-

vided by a number of vendors.

& An LC-MALDI spotter is significantly different to

the conventional MALDI spotter available as part

of the 2D PAGE-MS workflow for MALDI PMF.

LC-MALDI affords some attractive benefits to the researcher:

Fixation of samples to the MALDI target, allows

longer data collection and sample re-interroga-

tion at any time, removing the temporal con-

straints of the LC dimension.

An RPC separation will reduces the complexity

of the sample as discussed previously, which will

aid minimization of ion suppression.

Conservation of samples.

Probable separation of isobaric peptides (i.e. pep-

tides with the same apparent molecular weight),

which might have different hydrophobicities and

would therefore be resolved by LC.

Increased sequence coverage for higher confi-

dence in protein identification.

The LC-MALDI technique is a powerful adjunct to ESI based mass

spectrometers, with MALDI and ESI often providing complementary

protein identification information; Bodnar WM et al. 2004. This pub-

lication reported a “significant degree of overlap” (63%) was observed

between the proteins identified in the LC/ESI/MS/MS and LC/

MALDI/MS/MS data sets in the analysis of mammalian mitochron-

drial ribosomes. However, unique peptides and proteins were identi-

fied by each method; indicating that improved proteome coverage can

be obtained using a combination of these ionization techniques.

The technique has been applied to a number of challenging and

topical proteomics applications including the identification of mem-

brane proteins (Zhang et al. 2004); identification and quantification

Bodnar WM, Blackburn RK,

Krise JM, Moseley MA. J Am

Soc Mass Spectrom 14 (2003)

971–979.

Zhang N, Li N, Li L. J

Proteome Res 3 (2004)

719–727.

3 Mass Spectrometry222

using differential stable isotope labeling (Ji and Li 2005) and the

incorporation of high speed, high-resolution monolithic capillary col-

umns into the technique. In this publication, 384 unique peptides

were identified in a single 10 minute LC run from 2000 resolved com-

ponents (Chen et al. 2005).

3.1.2

Electrospray Ionization

Fenn et al. (1989); described the use of electrospray ionization (ESI)

for ionizing large biomolecules in 1989.

Electrospray (ESI) ions are produced at atmospheric pressure by

applying sample dissolved in solvent to a narrow capillary tube, which

is under the influence of an electric field. A potential difference is cre-

ated between the capillary and the inlet of the mass spectrometer gen-

erating a force extending the liquid to form a cone from the capillary

tip, from which a fine mist of droplets will emerge (Taylor cone; Tay-

lor 1964). The cone is formed as a result of repulsive coulombic forces

between the like charges. Subsequently, evaporation of the droplets

reduces droplet size whilst the charge on the droplet remains con-

stant. Eventually, the surface coulombic forces exceed the surface ten-

sion and the droplets fission into smaller droplets (the Rayleigh

limit). This process continues until nanometer-sized droplets are pro-

duced (Figure 3.4).

The charges are statistically distributed over the analyte’s potential

charge sites, enabling the formation of multiply charged ions. Each

multiply charged ion can be termed a charge state, and a distribution

of charge states is characteristic of large macromolecules during ESI

analysis.

& ESI produces predominantly multiply charged

ions.

Fig. 3.4: Schematic of ESI.

Ji C, Li L. J Proteome Res 4

(2005) 734–742.

Chen HS, Rejtar T, Andreev V,

Moskovets E, Karger BL. Anal

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Fenn JB, Mann M, Meng CK,

Wong SK, Whitehouse CM.

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A280 (1964) 383–397.

3.1 Ionization 223

This unique feature of ESI, multiple charging of analyte molecules,

yields an effectively reduced mass for the analyte of interest. Simply,

the observable mass range is lowered offering significant advantages

for the analysis of large molecular weight analytes, particularly pro-

teins. Figure 3.5 demonstrates the multiply charging of 150 KDa pro-

tein. With respect to proteins the resolution of each multiply charged

ion is insufficient to determine the number of charges the ion pos-

sesses, from which can be determined its true mass. Therefore, the

masses of at least two of the multiply charged ions in the envelope

are used in a deconvolution algorithm to give the mass of the whole

protein (lower panel).

Fig. 3.5: Characteristic multiple charging of a full-length protein

acquired using ESI. Upper panel: true mass of protein after deconvolution.

Lower panel: multiple-charged spectrum. Note the number of charge

states observed for this 150 kDa protein.

Note the mass range and the

number of charges (z value in

m/z) on the protein in the spec-

trum).

Its mass is known from the

spectrum, but without knowl-

edge of the charge state, the

real mass of the analyte

remains unknown.

3 Mass Spectrometry224

With respect to peptides, the charge state of the multiple-charged ion

can be readily determined due to the resolution capability of current

mass spectrometers, see Figure 3.6. The mass difference between the

adjacent

C and

C isotopes is indicative of the charge state. For a

single-charged ion, the mass difference between the

C and

C iso-

topes of a peptide isotopic envelope will be 1 Da. In the case of a dou-

ble-charged peptide ion (m/z = m/2) the mass difference between the

C and

C isotopes will be 0.5 Da, for a triple-charged peptide (m/z

= m/3) the mass difference between the

C and

C will be 0.33 Da

and so on. With knowledge of the charge state, an accurate mass of

the peptide can be determined.

Fig. 3.6: Characteristic multiple charging of tryptic peptides:

triple-charged neurotensin protein acquired using ESI.

Electrospray generates flow rates ranging from tens of microliters to

hundreds of microliters per minute. The resultant high consumption

rate was problematic for proteomic applications with respect to sensi-

tivity and analysis time (high amount of sample is consumed in a

short amount of time). In the early nineties, reductions in ESI low

rates were reported; sub-microliter flow rates using continuous infu-

sion affording significant increases in sensitivity (Emmett and

Caprioli 1994). Wilm and Mann reported the use of a nanospray ioni-

zation source. (Wilm and Mann 1996) This miniaturization of electro-

spray generated flow rates in the order of tens of nanoliters per min-

ute (typically 25–40 nL/min), by spraying the solvent from a narrow

3.2 Ion Separation 225

bore coated needle (sub 10 mm tip diameter). This was a crucial devel-

opment; delivering higher sensitivity and longer analysis times,

enabling multiple MS/MS experiments to be performed per run.

Though nanospray generates flows of 25–40 nL/min, nanoLC (at

least 75 mm i.d columns) only generates flow rates of 200 nL/min.

3.2

Ion Separation

A wide range of analyzers, compatible with both MALDI and ESI, are

used for proteomics applications

3.2.1

Time-of-Flight Analyzer

The time-of-flight (TOF) analyzer is a pulsed analyzer and as such is

routinely coupled with a MALDI ion source. (see Figure 3.7).

Fig. 3.7: Schematic of a reflectron TOF analyzer. This analyzer is

typically coupled with MALDI to perform peptide mass fingerprinting.

Mass measurement is determined by measuring the time-of-flight of

an ion in the analyzer region; the time-of-flight of an ion is propor-

tional to the square root of its mass/charge ratio, given a constant

accelerating voltage: Time of flight k

ﬃﬃﬃﬃﬃﬃﬃﬃﬃ

m=z

The linear TOF analyzer is the simplest form and exhibits the lower

performance with respect to resolution and mass accuracy. A linear

TOF instrument is incapable of resolving the

C and

C isotopes of

the peptide isotopic envelope across the range of the protein digest,

700–3500 Da, subsequently mass accuracy is poorer as the average

mass, not the mono-isotopic mass, tends to be used for database

searching.

Emmett MR, Caprioli RM. J

Am Soc Mass Spectrom 5

(1994) 605–613.

Wilm M, Mann M. Anal Chem

68 (1996) 1–8.

Thus, the smaller the molecule

the faster it will travel the

distance of the flight tube to

the detector.

3 Mass Spectrometry226

The performance of TOF analyzers with respect to resolution and

mass accuracy was vastly improved following two developments in

the 1990s: delayed extraction and incorporation of a reflectron.

Delayed extraction Resolution is improved by delaying the extraction

of ions from the source for a short period of time. Wiley and McLa-

ren, 1953; reported the method time lag focusing and noted the

increased resolution. This was revisited by Brown and Lennon, Vestal

et al. Whittal and Li in 1995 by applying it to MALDI. They observed

that by delaying the extraction of ions from the source, a much tighter

packet of ions arrive at the detector giving significantly higher resolu-

tion and mass accuracy.

Incorporation of a reflectron A reflectron (Mamyrin 2001) improves

resolution in two ways: (1) by acting as an ion mirror reversing the

trajectory of the ions in the flight tube, effectively increasing the

length of the flight tube; (2) by reducing an ion’s kinetic energy

spread. Ions of the same mass formed in the source can have differ-

ent kinetic energies when they leave the source depending upon their

position in the source when the accelerating voltage was applied, con-

sequently, ions of the same mass arrive at the detector at different

times, thus reducing resolution and mass accuracy. A reflectron can

accommodate these small differences in kinetic energy with the

result that ions of the same mass are better time-focused at the detec-

tor, greatly improving resolution and subsequently mass accuracy.

Delayed extraction coupled with a reflectron can routinely achieve

resolutions greater than 10,000 (FWHM; Figure 3.8). This level of

performance enabled MALDI-TOF to become established as the

principle method of protein ID in the 2D PAGE-MS workflow (Sec-

tion 1.7).

Reflectrons are incorporated as standard in most commercial TOF

mass spectrometers and the different configurations include a con-

ventional linear field reflectron, the curved field reflectron (Cornish

and Cotter, 1994) and the harmonic or quadratic field described by

Anderson et al. 1998.

Although a TOF analyzer is commonly coupled with a pulsed

MALDI source it has also been combined with electrospray, in the

ESI-TOF instrument (Boyle and Whitehouse 1992; Mirgorodskaya

et al. 1994; Verentchikov et al. 1994).

A reflectron TOF instrument is typically used in MS mode, though

a technique called post source decay (PSD) has been considered a

pseudo MS/MS technique (see Section 3.3.4.4).

Wiley WC , McLaren IH. Rev

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1050.

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trom 206 (2001) 251–266.

Protein database search confi-

dence using MALDI TOF PMF

data is improved with increased

mass accuracy

Cornish TJ, Cotter RJ. Rapid

Commun Mass Spectrom 8

(1994) 781–785.

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SC, Hoffman AD , Thomson S.

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3.2 Ion Separation 227

Fig. 3.8: Delayed extraction coupled with a reflectron can

routinely achieve resolutions greater than 10,000 (FWHM).

3.2.2

Triple Quadrupole Analyzer

The quadrupole analyzer consists of four parallel hyperbolic rods,

through which the gas phase ions have to achieve a stable trajectory.

The analyzer is operated by the application of a voltage (DC) and an

oscillating voltage (radio frequency, rf) to one pair of rods and DC vol-

tage of opposite polarity and rf voltage of different phase to the oppo-

site pair of rods. The alternating electric field helps to stabilize and

destabilize the passing ions. The ions traverse through the space

between the rods, and only at specific voltages applied to the rods will

certain m/z values be allowed to pass through the rods and reach the

detector. The voltages are scanned to allow a wide mass range to be

observed.

A single quadrupole analyzer has negligible benefits for proteomic

analysis, but if three quadrupoles are arranged in sequence, the sub-

sequent triple quadrupole analyzer coupled with ESI has been

demonstrated to be very useful for proteomics (von Haller et al. 2001)

This configuration is almost exclusively used to provide structural

information by tandem mass spectrometry (see Section 3.3.4). Here

the first and third quadrupoles operate as the mass filters described

above, with the second quadrupole operating as a collision cell (where

the structural information is produced). The analyzer can be operated

von Haller PD, Donohoe S,

Goodlett DR, Aebersold R,

Watts JD. Proteomics 1 (2001)

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3 Mass Spectrometry228

in three different ways to perform MS/MS experiments (see Section

3.3.4.1) and is typically used in MS/MS mode (see Figure 3.9).

Fig. 3.9: Schematic of a triple quadrupole analyzer. Coupled with

ESI, it can perform a range of MS/MS techniques including product

ion, precursor ion and neutral loss scans.

3.2.3

Quadrupole Ion Trap

The quadrupole ion trap is based on the same theory as the quadru-

pole analyzer, with the quadrupole field generated within a three

dimensional trap. The ion trap itself is filled with helium and com-

prises a ring electrode and two end cap electrodes, creating the three

dimensional electric field by applying a large RF voltage to the ring

electrode. The orbiting motion of the ions in the trap is governed by

the large rf voltage and the cooling effects of collisions with the

helium gas (the helium gas reduces the kinetic energy of the ions

and helps focus the ions in the center of the trap). A mass spectrum

is acquired by sequentially ejecting fragment ions from low m/z to

high m/z. This is performed by scanning the rf voltage to make ion

trajectories sequentially become unstable, the mass selective instabil-

ity mode developed by Stafford et al. (1984). Ions are ejected and

detected at the detector (see Figure 3.10).

Fig. 3.10: Schematic of the fundamental operation of a 3D ion

trap analyzer. Commonly coupled with ESI for proteomics applications.

Stafford GC, Kelley PE, Syka

JEP, Reynolds WE, Todd, JFJ.

Int J Mass Spectrom Ion Proc

60 (1984) 85–98.

3.2 Ion Separation 229

A product ion MS/MS spectrum is obtained via resonance excitation.

The precursor ion is isolated by the application of a waveform signal

to the endcap electrodes. Fragmentation of the precursor ion is

caused by the application of a small voltage across the endcap electro-

des and collisions with helium gas. The resultant fragmentation is

similar to that observed from a triple quadrupole mass spectrometer;

a low-energy collisional regime with fragmentation predominantly at

the peptide amide bonds (see Section 3.3.4.1). A drawback of the

instrument for the acquisition of product ion MS/MS spectra is that

the fragment ions in the lower third of the mass range (approximately

1/3

of the mass of the precursor ion) will not be detected.

An additional advantage of quadrupole ion traps and FT-ICR MS

(see Section 3.2.7) is the unique ability to perform multiple stages of

mass spectrometry (MS

) (see Section 3.3.4.5). For a detailed review

see Jonscher and Yates (1997).

The performance characteristics of a quadrupole ion trap make it a

highly attractive instrument for proteomic applications, particularly

for tandem mass spectrometry applications, either interfaced with

nanospray (Hoess et al. 1999) LC-MS/MS (Ho et al. 2002) 2DLC-

MS/MS (Washburn et al. 2001) or MALDI (Qin et al. 1996). The ion

trap is predominantly used in MS/MS mode.

Developments in ion trap technology have delivered the linear

quadrupole ion trap, where ions are trapped in a two-dimensional

quadrupole field, instead of a three-dimensional quadrupole field.

Such a configuration affords significant benefits for protein ID and

characterization including rapid cycle time which enables more infor-

mation to be obtained from a sample in less time with high sensitiv-

ity (Yates et al. 2006). The linear ion traps also offer additional tandem

mass spectrometry functionality (Macek et al. 2006). In addition, to

, commercial ion trap systems are available that can perform a

novel fragmentation technique, electron transfer dissociation or ETD

(Thermo LTQ and Bruker Esquire HCT Ultra), see section 3.3.4.6.

ETD, a peptide and protein fragmentation technique that preserves

modifications, has found applications in PTM analysis such as phos-

phorylation or glycosylation and in top down proteomics. The techni-

que extends the capability of the user with respect to protein sequen-

cing and identification of type and location of various PTMs. The

common approach is to use both collision induced dissociation and

ETD, generating non-redundant sequence information.

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