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B5 Proteomics and Applications PDF
B5 Proteomics and Applications PDF
development pipeline
B5
René Houtman and Ian Humphery-Smith
(positive) anode until they reach their pI. At this forming chemicals. However a major improvement
point the residue’s charge is neutralized by proton of the technique was accomplished with the intro-
uptake from its environment resulting in an arrest of duction of immobilized pH gradients (IPG) in the
migration.Alternatively, at a pH above below their pI, late 1980s. The latter, formed by ampholytes cross-
amino acids (β) behave as proton acceptors result- linked to a plastic carrier strip by acryl amide, result-
ing in a positive charge. Accordingly, in an electric ed in enhanced resolution,improved reproducibility,
field they move to the cathode until their pI is higher loading capacity for preparative gels and pos-
reached. The pI of a protein is determined by the sibility of separation of basic proteins under equilib-
average of the pIs of the residues of which it consists. rium conditions [5], but is usually practiced in the
Proteins with different amino acid compositions presence of ampholytes and thus referred to as
therefore have different pIs. Originally, IEF of proteins ‘mixed-bed’ electrophoresis.
was performed in rod-shaped polyacrylamide gels Following IEF, proteins are separated with respect
with mixed-in synthetic ampholytes, pH gradient- to mass within a polyacrylamide sieving matrix,
Traditional proteomics 215
FIGURE 4
Quantitative mass spectrometry-based analysis of protein expression by stable isotope labeling. Comparison of pro-
tein abundance between two samples is performed by labeling the proteins of the samples with different mass tags.
This labeling is mainly done A. metabolically, B. chemically, e.g., isotope-coded affinity tagging (ICAT, see also D.) or
C. proteolytically. Subsequently, labeled samples are mixed, digested and subjected to mass spectrometry. In the result-
ing spectrum, identical peptides of the different samples are separated by the mass difference of the two isotopes and
the ratio of their relative abundance in the spectrum reflects the ratio of protein expression between the two samples.
mass of an amino acid and a PTM as listed in lar and regulatory proteins; however, only highly
Table 2. expressed proteins are detected and identified with
current 2DE protocols [2, 8, 9].The tendency of 2DE
to detect only highly expressed proteins is caused by
2DE drawbacks the intrinsic limitations in dynamic range of various
stages of the technique on one hand and the enor-
Although 2DE is unsurpassed in its resolving power mous dynamic range of protein expression level
for separation of complex protein mixtures, it is not across a PROTEOME on the other.
without difficulties and shortcomings. Low-abun- The dynamic range of detection by classical
dance proteins are probably the most interesting methods such as Coomassie Blue (low sensitivity) or
ones from the point of view of understanding cellu- silver staining (non-linear at higher protein concen-
220 Proteomics and applications within the drug development pipeline
Alanine A 71 44
Arginine R 156 129
Asparagine N 114 87
Aspartic Acid D 115 88
Cysteine C 103 76
Glutamic Acid E 129 102
Glutamine G 128 101
Glycine Q 57 30
Histidine G 137 110
Isoleucine H 113 86
Leucine I 113 86
Lysine L 128 101
Methionine M 131 104
Phenylalanine F 147 120
Proline P 97 70
Serine S 87 60
Threonine T 101 74
Tryptophan W 186 159
Tyrosine Y 163 136
Valine V 99 72
trations) is too narrow and therefore fluorescent tion of all proteins during sample preparation virtu-
staining methods with new probes with enhanced ally impossible. To reduce complexity, samples can
SENSITIVITY and dynamic range have been developed be pre-fractionated for specific subsets of proteins by
and are becoming increasingly popular. In addition, affinity enrichment and application of specific
the number of proteins that can be separated practi- extraction buffers and methods. Still, hydrophobic
cally by 2DE is limited by the physical dimensions of proteins such as membrane receptors often precipi-
the gel itself. One solution to enhance resolution is tate during IEF, which prevents transfer into the SDS-
separation of a protein sample on multiple 2DE gels PAGE gel and are thus lost during separation.
of consecutive partially overlapping narrow-range 2DE also suffers from high variation that is intro-
IPGs. Subsequently, the images of generated gels are duced during sample preparation, protein loading
combined in silico to construct so-called ‘ZOOM GELS’ and the complex nature of the staining procedures
[5, 10]. employed. In order to obtain statistically significant
The high variety in protein expression levels is quantitative data from which valid conclusions
illustrated by the fact that 90% of the PROTEOME of a regarding biology can be drawn, it is necessary to
typical cell is made up of only 10% of the produce a number of replicate gels from the same
10,000–20,000 different protein species. An addition- sample [11–13]. Furthermore, qualitative and quanti-
al factor that adds to this complexity is the large vari- tative analysis of 2DE gels performed by dedicated
ety of biochemical characteristics (hydrophobicity, software is still difficult to automate and requires a
charge, pI, etc.) of proteins, which makes solubiliza- high degree of human intervention.The latter step is
Traditional proteomics 221
therefore time consuming and currently the rate-lim- by linearly altering the amplitude of the radio fre-
iting factor in 2DE-based PROTEOME analysis. These quency applied to the ring electrode so as to elute a
shortcomings have been ostensibly overcome and particular ion series from the trap, after which they
fully automated image analysis is becoming a long- are analyzed by TOF.
awaited reality in the discipline.As with any process, The MudPIT method was shown to be effective in
when one bottleneck has been overcome, the goal- identification of proteins with a wide range of bio-
posts are transposed elsewhere. Independent of chemical properties and expression levels, including
method employed, the major challenge for PRO- low-abundance proteins [14,15].Although MudPIT is
TEOMICS is that of total proteomic coverage, which easy to automate and harbors the appropriate reso-
remains low, except in microbes. lution and dynamic detection range needed to man-
age the complexity of global PROTEOME analysis, it
will only generate qualitative data.This is a restriction
Qualitative MS-based proteomics of all MS-based techniques because the relative
abundance of a peptide in the spectrum depends on
Because of the limitations of 2DE, other techniques its biochemical make-up and subsequent ability to
have been developed for qualitative PROTEOME analy- become ionized and is not necessarily correlated
sis based on liquid separations coupled to on-line with its concentration in the sample, i.e., relative ion
mass spectrometry. In general, complex mixtures of intensity at the detector may be significantly differ-
proteins,or peptides,are separated by microcapillary ent from that entering the mass spectrometer.
format liquid chromatography, capillary elec-
trophoresis or hybrids thereof prior to ESI and subse-
quent mass analysis by MS/MS. This approach using Quantitative MS-based proteomics
one-dimensional separation is sufficient for the
analysis of low-complexity samples; however,its reso- Several alternative methods have been proposed for
lution is by far inferior to that of 2DE.To date,no stud- relative quantification of protein expression between
ies have been conducted on multiple analyses of the paired samples, of which one has been subjected to
same sample set, but initial indications are that vari- stable isotope labeling, which can be achieved by
ance due to technology and biology will mean that several methods (Fig. 4).
large numbers of replicates will be necessary to
allow valid conclusions due to significant popula- Metabolic labeling
tion variance in both test and control groups.
One approach to obtain a higher order of resolu- In this approach (Fig.4A),two populations of cells or
tion of separation prior to MS is termed multidimen- organisms are grown in parallel, one on a normal
sional protein identification technology (MudPIT) source of nutrition and the other on a source deplet-
[14]. This orthogonal combination of cation ed or enriched for an isotope of N, C or H [16–18].
exchange and reverse-phase chromatography con- This process is referred to as stable isotope labeling
sists of sequential step-elutions (~15) from global with amino acids during culture,or SILAC [19].Next,
styptic peptides from a strong cation exchange resin protein samples from both populations are mixed,
with slow ethanol gradients inserted between each digested and analyzed in parallel by MS. The incor-
salt elution step. After ESI, eluted peptides are ana- porated isotope induces a mass change reflected by
lyzed using a quadrupole ion trap (QIT).This instru- a characteristic shift in the resultant MS spectrum.
ment consists of two hemispherical end-cap elec- The mean ratio in relative ion intensity of correspon-
trodes,spatially separated by ring electrodes,illustrat- ding peaks is employed as an indication of that of
ed in Figure 3. The trapping potential, AC voltage, the relative difference in protein expression
applied to the end-cap electrodes, is used to confine between the two populations (Fig. 4E). Unfortunate-
ion species with respect to their respective m/z ratio ly, peptides from the same protein show much varia-
for subsequent analysis.A mass spectrum is obtained tion with respect to the estimates of protein abun-
222 Proteomics and applications within the drug development pipeline
dance derived from ion intensity measures, due to Mass coded abundance tagging
differences in a peptide’s overall charge and associ-
ated willingness to enter the gas phase and ‘fly’ in To avoid problems associated with poor quantifica-
the MS. tion derived from estimates based on ion intensity
measures, mass coded abundance tagging (MCAT)
Isotope-coded affinity tagging has been introduced [22].The methodology is based
on guanidination of C-terminal lysine residues on
Another method is isotope-coded affinity tagging tryptic peptides, corresponding elution times, direct
(ICAT) [20] (Fig. 4B) or variants thereof based upon peptide identification by MS and a predictable mass
isotope tagging of sulphydryl groups, amino groups, change of 21 m/z units. Here, however, ACCURATE pro-
active sites for serine & cysteine hydrolases, phos- tein quantification can be achieved from a recon-
phate ester groups, and N-linked carbohydrates. The structed ion chromatogram for comparison of ‘areas
ICAT reagents (Fig. 4D), consist of a protein-(cys- under the curve’ for differential screening. The latter
teine) reactive group,a linker region and a biotin tag. is somewhat limited by the resolution afforded by
The linkers are composed of either 8 deuterium (d8, the liquid chromatography system. Another variant
heavy reagent) or 8 hydrogen (d0, light reagent) of MCAT is absolute quantification (AQUA) internal
atoms, resulting in an 8Da mass difference between standard peptides [23].
corresponding peaks in the MS spectrum.A reduced
protein sample from one population is derivatized, Proteolytic labeling
on cysteine,with the isotopically heavy version of the
ICAT reagents, while the other is derivatized with the A third labeling method is performed during protein
isotopically light variant. Derivatized samples are digestion (Fig.4C).Two samples are separately digest-
mixed and subjected to digestion, after which the ed in either H216O or H218O.The oxygen atom derived
mix is enriched for cysteine-containing peptides by from the aqueous solvent is incorporated into the
affinity purification using the biotin tag of the ICAT newly formed C-terminus of each peptide, providing
linker. This results in an approximate tenfold reduc- an EPITOPE tag for relative quantification [23]. Differ-
tion in sample complexity. One of the weaknesses of ential resolution can be achieved in a similar man-
this method is the fact that 1 out of 7 proteins does ner by incorporation of 15N [24].
not contain any cysteine and thus does not react
with the ICAT reagents.These proteins are lost during
affinity purification using biotin and therefore Array-based proteomics
missed in the analysis. Furthermore, the ICAT
reagents are relatively large molecules compared MICROARRAYS provide the potential for determining
with the peptide to which they are attached and thousands of different binding events in a single
thereby influence chromatographic separation prior experiment in a massively parallel fashion. The
to ionization and peptide ionization itself, which genomic revolution of the last decade which has
complicates data interpretation. A detailed compari- delivered information on whole organisms at the
son of 2DE and ICAT technologies has shown level of DNA sequence and transcriptome analysis
healthy complementarity of these techniques,where- has been based notably on parallelization, miniatur-
by together enhanced proteomic coverage was ization and automation – all of which become possi-
achieved with the latter demonstrating a bias for ble in a protein biochip environment. Usually, a num-
high-Mr proteins and quantification based on the ber of different molecules with distinct binding char-
sum of the protein species, while the former showed acteristics, CAPTURE MOLECULES or ligands, are
improved resolution of low-Mr proteins, post-transla- immobilized in a grid-like fashion on a solid SUB-
tionally modified polypeptides, processed polypep- STRATE, the array.The latter is incubated with an ANA-
tides and identified cysteine-free proteins, otherwise LYTE solution containing the molecules of interest,
invisible to ICAT [21]. TARGETS.Detection of binding is usually performed by
Array-based proteomics 223
face density onto spots that have increased spot but the signal density will increase for smaller spots.
size. With increasing spot size, the total amount of Below a certain spot size, the signal density
CAPTURE MOLECULES in the assay increases,as does the approaches an optimum (ambient ANALYTE condi-
sum of the obtained signal in the spot. The signal tions, unlimited amount of TARGET) and will stay
density, however, starts to decrease with increasing approximately constant with any further decrease of
spot size because the amount of TARGET starts to spot size.Therefore the highest signal intensities and
become a limiting factor.The capture process leads optimal signal-to-noise ratios can be achieved in
to a significant reduction of TARGET concentration in small spots. Small spot size also allows for dramatic
solution and at the same time the probe-TARGET com- parallelization on a low-cost biochip format.
plexes are distributed over a larger area. As a result Furthermore, when conducted in conjunction with
the maximal signal that can be obtained from any grating-coupled surface plasmon resonance, real-
spot in the spot is decreased. Decreasing the spot time association and dissociation constants can be
size will decrease the overall signal per MICROSPOT acquired in parallel so as to provide still greater
Summary 225
insight into the dynamics of biomolecular interac- with the progression, prognosis and early detection
tions. of disease and are also being applied to patient
To date, protein biochips have been employed to cohorting during drug trials, i.e., pharmacopro-
examine a wide variety of assay types, see Figure 7. teomics. Protein arrays hold equally great potential
These include detection of interactions between to improved lead development and optimization as
antibody-antigen; protein-protein; protein-nucleic a means of verifying TARGET SPECIFICITY in the presence
acid, protein-small molecule, membrane-bound of numerous potential recombinant binders and
receptors and TARGET,domain screening,and analysis thereby working towards reducing adverse drug
of enzymatic function. In addition, a parallelized for- effects as a direct spin-off from increased knowledge
mat has been adopted for a variety of other applica- of the human genome.
tions in PROTEOMICS.These applications include tissue
arrays, EPITOPE mapping via peptide arrays, reverse
arrays for the examination of naturally occurring pro- Summary
tein isoforms found in cellular extracts as eluted
from liquid chromatography,cellular arrays for bioas- Here,we have set out to demonstrate that PROTEOMICS
says, and immobilization of various chromatography in its various forms has demonstrated its utility
affinity capture reagents. The latter are now being across the entire drug development pipeline from
used extensively to seek out biomarkers associated initial TARGET discovery and validation through to
226 Proteomics and applications within the drug development pipeline
monitoring drug responses during clinical trials. In An Introduction to Mass Spectrometry: http://www.ast-
recent times, proteomic techniques are also increas- bury.leeds.ac.uk/Facil/MStut/mstutorial.htm (Acces-
ingly making their impact felt in the very last step in sed December 2004)
this pipeline, namely process validation during the Swiss Proteomics Society (SPS): http://www.swisspro-
production phase.This more generalized impact has teomicsociety.org/links.html (Accessed December
only become possible as a result of improved repro- 2004)
ducibility,sensitivity,user-friendliness and throughput Proteomics – Spectroscopic Applications in Proteomics:
of the techniques employed. The next phase of http://www.spectroscopynow.com/Spy/basehtml/Sp
genomics is now generally hailed as focusing on yH/1,1181,10-4-0-0-0-directories_new-0-0,00.html
PROTEOMICS.These techniques are evolving rapidly to (Accessed December 2004)
meet the demands of the scientific and pharmaceu- North Carolina Genomics & Bioinformatics Consortium,
tical communities. Such specialized approaches as LLC: http://www.ncgbc.org/resources/links/proteo-
chemoproteomics are likely to usher in still further mics-links.cfm (Accessed December 2004)
advances in global screening of biomolecule bind- http://www.hupo.org/ (Accessed December 2004)
ing to therapeutic agents and high-throughput char-
acterization on line via mass spectrometry of these
binders.The results of such analyses can then be fed References
to in silico analysis so as to better understand the
shared structural motifs and domains that have given 1 Varshavsky A (1996) The N-end rule: functions, myster-
rise to drug binding across a wide spectrum of cellu- ies, uses. Proc Natl Acad Sci USA 93: 12142–12149
lar components. In such a manner, PROTEOMICS data 2 Gygi SP, Rochon Y, Franza BR, Aebersold R (1999) Cor-
are likely to be seen increasingly to actually drive relation between protein and mRNA abundance in
many aspects of future drug development and yeast. Mol Cell Biol 19: 1720–1730
improved safety of therapeutic agents, be they tradi- 3 Futcher B, Latter GI, Monardo P, McLaughlin CS, Garrels
tional small molecules or biomolecules. JI (1999) A sampling of the yeast proteome. Mol Cell
Biol 19: 7357–7368
4 O’Farrell PH (1975) High resolution two-dimensional
Selected Readings electrophoresis of proteins. J Biol Chem 250: 4007–
4021
Aebersold R,Mann M (2003) Mass spectrometry-based pro- 5 Gorg A, Obermaier C, Boguth G, Harder A, Scheibe B,
teomics. Nature 422: 198–207 Wildgruber R,Weiss W (2000) The current state of two-
Phizicky E, Bastiaens PI, Zhu H, Snyder M, Fields S (2003) dimensional electrophoresis with immobilized pH
Protein analysis on a proteomic scale. Nature 422: gradients. Electrophoresis 21: 1037–1053
208–215 6 Humphery-Smith I,Ward M (2000) Proteome research:
methods for protein characterization. In: S Hunt, F
Livesey F (eds): Functional Genomics. Practical
Recommended Websites Approach. Oxford University Press, New York, 197–241
7 Cordwell SJ, Wilkins MR, Cerpa-Poljak A, Gooley AA,
Web resources for protein scientists: http://www.proteinso- Duncan M, Williams KL, Humphery-Smith I (1995)
ciety.org/docs/WWWResources.html (Accessed De- Cross-species identification of proteins separated by
cember 2004) two-dimensional gel electrophoresis using matrix-
IonSource: http://ionsource.com/ (Accessed December assisted laser desorption ionisation/time-of-flight mass
2004) spectrometry and amino acid composition. Elec-
ExPASy Proteomics Server: http://www.expasy.org/ (Ac- trophoresis 16: 438–443
cessed December 2004) 8 Gygi SP, Corthals GL, Zhang Y, Rochon Y, Aebersold R
Proteomics Interest Group (ProtIG): http://proteome. (2000) Evaluation of two-dimensional gel elec-
nih.gov/links.html (Accessed December 2004)
References 227
trophoresis-based proteome analysis technology. Proc 20 Gygi, SP, Rist B, Gerber SA, Turecek F, Gelb MH, Aeber-
Natl Acad Sci USA 97: 9390–9395 sold R (1999) Quantitative analysis of complex protein
9 Velculescu VE, Zhang L, Zhou W, Vogelstein J, Basrai mixtures using isotope-coded affinity tags.Nat Biotech-
MA, Bassett DE Jr, Hieter P, Vogelstein B, Kinzler KW nol 17: 994–999
(1997) Characterization of the yeast transcriptome. 21 Schmidt F, Donahoe S, Hagens K, Mattow J, Schaible
Cell 88: 243–251 UE, Kaufmann SH, Aebersold R, Jungblut PR (2003)
10 Hoving S, Gerrits B,Voshol H, Muller D, Roberts RC, van Complementary analysis of the Mycobacterium tuber-
Oostrum J (2002) Preparative two-dimensional gel culosis proteome by two-dimensional electrophoresis
electrophoresis at alkaline pH using narrow range and isotope coded affinity tag technology. Mol Cell
immobilized pH gradients. Proteomics 2: 127–134 Proteomics 3: 24–42
11 Voss T, Haberl P (2000) Observations on the repro- 22 Cagney G,Emili A (2002) De novo peptide sequencing
ducibility and matching efficiency of two-dimensional and quantitative profiling of complex protein mixtures
electrophoresis gels: consequences for comprehen- using mass-coded abundance tagging. Nat Biotechnol
sive data analysis. Electrophoresis 21: 3345–3350 20: 163–170
12 Quadroni M, James P (1999) Proteomics and automa- 23 Gerber SA, Rush J, Stemman O, Kirschner MW, Gygi SP
tion. Electrophoresis 20: 664–677 (2003) Absolute quantification of proteins and phos-
13 Houtman R, Krijgsveld J, Kool M, Romijn EP, Redegeld phoproteins from cell lysates by tandem MS. Proc Natl
FA, Nijkamp FP, Heck AJ, Humphery-Smith I (2003) Acad Sci USA 100: 6940–6945
Lung proteome alterations in a mouse model for non- 24 Munchbach M, Quadroni M, Miotto G, James P (2000)
allergic asthma. Proteomics 3: 2008–2018 Quantitation and facilitated de novo sequencing of
14 Washburn MP, Wolters D, Yates JR 3rd (2001) Large- proteins by isotopic N-terminal labeling of peptides
scale analysis of the yeast proteome by multidimen- with a fragmentation-directing moiety. Anal Chem 72:
sional protein identification technology. Nat Biotech- 4047–4057
nol 19: 242–247 25 Humphery-Smith I (2003) Protein Biochips and Array-
15 Wolters DA,Washburn MP,Yates JR 3rd (2001) An auto- based Proteomics. Dekker, New York
mated multidimensional protein identification tech- 26 Masseyeff RF (1991) Standardization of immunoas-
nology for shotgun proteomics. Anal Chem 73: says. Ann Ist Super Sanita 27: 427–436
5683–5690 27 Ekins RP (1989) Multi-analyte immunoassay. J Pharm
16 Weckwerth W, Willmitzer L, Fiehn O (2000) Compara- Biomed Anal 7: 155–168
tive quantification and identification of phosphopro- 28 Templin MF, Stoll D, Schrenk M,Traub PC,Vohringer CF,
teins using stable isotope labeling and liquid chro- Joos TO (2002) Protein microarray technology. Trends
matography/mass spectrometry. Rapid Commun Mass Biotechnol 20: 160–166
Spectrom 14: 1677–1681
17 Berger SJ, Lee SW, Anderson GA, Pasa-Tolic L, Tolic N,
Shen Y,Zhao R,Smith RD (2002) High-throughput glob-
al peptide proteomic analysis by combining stable
isotope amino acid labeling and data-dependent mul-
tiplexed-MS/MS. Anal Chem 74: 4994–5000
18 Krijgsveld J, Ketting RF, Mahmoudi T, Johansen J, Artal-
Sanz M,Verrijzer CP, Plasterk RH, Heck AJ (2003) Meta-
bolic labeling of C. elegans and D. melanogaster for
quantitative proteomics. Nat Biotechnol 21: 927–931
19 Ong SE, Blagoev B, Kratchmarova I, Kristensen DB,
Steen H, Pandey A, Mann M (2002) Stable isotope
labeling by amino acids in cell culture,SILAC,as a sim-
ple and accurate approach to expression proteomics.
Mol Cell Proteomics 1: 376–386