Saudi Pharmaceutical Journal (2010) 18, 59–73

King Saud University

Saudi Pharmaceutical Journal

www.ksu.edu.sa
www.sciencedirect.com

REVIEW ARTICLE

Separation of biological proteins by liquid chromatography

Imran Ali a,*, Hassan Y. Aboul-Enein b, Prashant Singh c, Rakesh Singh d,
Bhavtosh Sharma a

a
Department of Chemistry, Jamia Millia Islamia (Central University), New Delhi 110 025, India
b
Pharmaceutical and Medicinal Chemistry Department, Pharmaceutical and Drug Industries Research Division,
National Research Centre, Dokki, Cairo 12311, Egypt
c
Department of Chemistry, Dayanand Anglo Vaidic Post Graduate (D.A.V.P.G.) College, Dehradun 248 001, India
d
Department of Chemistry, Dayanand Brijendra Swaroop Post Graduate (D.B.S.P.G.) College, Dehradun 248 001, India

Received 28 May 2009; accepted 3 November 2009

Available online 13 February 2010

KEYWORDS Abstract After the success of human genome project, proteome is a new emerging field of bio-
Chirality; chemistry as it provides the knowledge of enzymes (proteins) interactions with different body
Gene; organs and medicines administrated into human body. Therefore, the study of proteomics is very
Liquid chromatography; important for the development of new and effective drugs to control many lethal diseases. In pro-
Nano detection; teomics study, analyses of proteome is essential and significant from the pathological point of views,
Proteomics; i.e., in several serious diseases such as cancer, Alzheimer’s disease and aging, heart diseases and also
Preparation for plant biology. The separation and identification of proteomics is a challenging job due to their

Abbreviations: ACN, acetonitrile; AIEC, anion exchange chromatography; CEC, capillary electro-chromatography; CIEF, capillary isoelectric
focusing; CSF, cerebrospinal fluid; 2D-nano LC, two-dimensional nano liquid chromatography quadrupole; Q-TOFMS/MS, time-of-flight
tandem-mass spectrometry; EC, electro-chromatography; ESI-LC–MS, electrospray ionization liquid chromatography–mass spectrometry; FA,
formic acid; FLP, FMRF amide-like peptide; GPI-APs, glycosylphosphadylinositol anchored proteins; GSH, glutathione stimulating hormone;
GSTs, glutathione-S-transferase isoenzyme; HFBA, heptafluorobutyric acid; HPLC, high performance liquid chromatography; ICAT, isotope
coded affinity tag; IEF-SEC, isoelectrofocussing size-exclusion chromatography; IMCD, inner medullary collecting duct; LC–MS, liquid
chromatography–mass spectrometry; LC-Q-TOF, liquid chromatography-quadrupole time-of-flight tandem mass; MS/MS, spectrometry; LC-dual
ESI, liquid chromatography dual electrospray ionization-Fourier transform; FT-ICR-MS, ion cyclotron resonance-mass spectrometry; MALDI-
TOF, matrix-assisted laser desorption/ionization-time-of flight; MFGM, milk fat globule membranes; MMA, mass measurement accuracy; MPC,
mesenchymal progenitor cell; NLFs, Nasal lavage fluids; NLP, neuropeptide like protein; PC2, prohormone convertase-2; PS II, photosystem II;
RPLC, reversed phase liquid chromatography; SCX, strong cation exchange; SEC, size-exclusion chromatography; TFA, trifluoroacetic acid; TIC,
total ion current; TRAF, tumor necrosis factor receptor
* Corresponding author.
E-mail address: imran.chem@yahoo.com (I. Ali).

1319-0164 ª 2010 King Saud University. All rights reserved. Peer-

review under responsibility of King Saud University.
doi:10.1016/j.jsps.2010.02.001

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60 I. Ali et al.

complex structures and closely related physico-chemical behaviors. However, the recent advances in
liquid chromatography make this job easy. Various kinds of liquid chromatography, along with dif-
ferent detectors and optimization strategies, have been discussed in this article. Besides, attempts
have been made to include chirality concept in proteomics for understanding mechanism and med-
ication of various disease controlled by different body proteins.
ª 2010 King Saud University. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
1.1. Separation methods for proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
1.2. Reversed phase high performance liquid chromatography. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
1.3. Affinity high performance liquid chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
1.4. Gel permeation high performance liquid chromatography. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
1.5. Ligand exchange high performance liquid chromatography. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
1.6. Capillary high performance liquid chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
1.7. Comparison of various chromatographic methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
1.8. Chirality and chirality and protomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
2. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71

1. Introduction chromatography, along with different detectors and optimiza-

tion, have been presented in this article.
Each cell produces thousands proteins in living organisms and a
set of them is called as proteome and unlike genome, the prote- 1.1. Separation methods for proteins
ome differs from cell to cell (Garcia et al., 2004). After success of
human genome project, proteome is a new emerging field of bio- Since the introduction of liquid chromatography in 1980 it has
chemistry as it provides the knowledge of enzymes (proteins) become very popular in analytical science but its applications
interactions with different body organs and medicines, adminis- came into practice in last decade. The nano detection makes
trated into human body. Therefore, the study of proteomics is these chromatographic techniques more useful in proteomic
very important for the development of new and effective drugs research. Various kinds of liquid chromatography methods
to control many lethal diseases. In proteomics study, analyses used in proteomic research are reversed phase, affinity, gel per-
of proteome are very important and significant from the patho- meation, ligand exchange and capillary liquid chromatogra-
logical point of views, i.e., in several serious diseases such as can- phies, which are discussed in this article.
cer (Le Naour et al., 2006; Vasilescu et al., 2005; Righetti et al.,
2005; Drew et al., 2005), Alzheimer’s disease and aging (Mon- 1.2. Reversed phase high performance liquid chromatography
tine et al., 2006), heart diseases (Banfi et al., 2006) and plant biol-
ogy (Glinski and Weckwerth, 2006). The separation and Reversed phase high performance liquid chromatography is
identification of proteomics is a challenging job due to their the most popular mode of chromatography due to its wide
complex structures and closely related physico-chemical behav- range of applications because of the availabilities of various
iors. However, literature indicated the successful use of liquid mobile and stationary phases. The on-line coupling of this
chromatography in this concern. Most effectively used kinds technique with sample preparation and detection units; spe-
of chromatography are liquid chromatography–mass spectrom- cially MS; makes it ideal technique in proteomics research.
etry (LC–MS) (Neverova and Van Eyk, 2005; Hortin et al., Nowadays, microchip based instruments are available to
2006), nano-reversed phase liquid chromatography (nano- achieve this difficult task.
RPLC) (Wang et al., 2005; Tyan et al., 2006) and ion exchange Some important separations of proteomics using RP-HPLC
chromatography (Lecchi et al., 2003). Platelets, having no nu- are discussed and analyzed critically. Yuan and Zhao (2001)
cleus in cells, are valuable to study hemostasis, thrombosis reported that multidimensional liquid chromatography cou-
and heart diseases. The proteins present in platelets have been pled with tandem-mass spectrometry has wide range of appli-
studied by multidimensional liquid chromatography followed cations in proteomics. Liang et al. (2006) quantified a group of
by mass spectrometry (Garcia et al., 2005). Over last few years, 1600 gene products into 997 protein families with 830 mem-
the proteomic analysis reveals that it requires the combination brane or membrane-bound proteins in normal and malignant
of on-line sample preparation and analytical methods due to breast cancer cells of a patient using nano-electrospray LC–
the diversity and complexity in proteomics structures. In view of MS/MS method. Crugliano et al. (2007) applied liquid chro-
these facts, attempts have been made to review the role of liquid matography with tandem-mass spectrometry for the analysis
chromatography in proteomics study. Various kinds of liquid of proteome of transfected HeLA cell lines having three clear
Separation of biological proteins by liquid chromatography 61

single amino acid changes in a nuclear phosphoprotein, i.e., Babusiak et al. (2007) reported 55 proteins including pepti-
BRCA1 protein. The authors reported that Met1775Arg and dase, ion channels, cycloskeletal proteins, enzymes of carbohy-
the Trp1837Arg did not show effective changes in comparison drate metabolism, regulatory enzymes etc. using PepMapTM
to cells transected having wild type BRCA1 cDNA and only C18 column (0.3 mm · 250 mm). Various proteins identified
BRCA1-Ser1841 Asn mutation creates effective changes in by LC MS/MS. Carlsohn et al. (2006) performed a nano-liquid
proteomic pattern in breast cancer patients. Sapra et al. chromatography Fourier transform-ion cyclotron response
(2006) reported a nano-LC–MS method for the proteomic mass spectrometry (nano-LC FT-ICR MS) analysis of the out-
analysis of two murine macrophages cell lines (J774.1A and er membrane protein of Helicobacter pylori, a human gastric
RAW 264.7), which were treated with Bacillus anthracis lethal pathogen which can create duodenal ulcers, gastric cancer dis-
toxin (LeTx) in anthrax infection. The authors identified five eases, using (17 cm · 50 lm i.d.) fused silica column packed
proteins as ATP synthase b-subunit, b-actin, Hsp 70, vimentin, with 3 lm ReproSil-Pur C18-AQ porous C18-bonded particles
and Hsp60 homolog, which were unregulated in above cell and identified 60 membrane associated proteins including (out-
lines. Pan et al. (2006) performed a quantitative neuropeptido- er membrane protein) Omp11 and BabA proteins in each
mic study for activity of prohormone convertase-2 (PC2) in strain. The authors reported that the fragmentation efficiency
processing of hypothalamic neuropeptides and reported 53 in the ion trap of the nano-LC FT-ICR MS and MS/MS anal-
neuropeptides or other peptides originating from 21 proteins ysis are more reproducible; Fig. 1. Seshi (2006) reported that
viz. proenkephalin, proopiomelanocortin, prodynorphin, 80 of 712 proteins in mesenchymal progenitor cell proteome
protachykinin A and B, procholecystokinin, promelanin-con- create 5258 of 10506 detected peptides. Few represented
centrating hormone, proneurotensin, proneuropeptide Y, pro- mesenchymal progenitor cell (MPC) proteins create a large
vasopressin, pronociceptin/orphanin, prothyrotropin-releasing number of MPC peptides, which are shown in Fig. 2. A com-
hormone, cocaine, amphetamine-regulated transcript, chro- parative study of peptides of different Caenorhabditis elegans
mogranin A and B, secretogranin II, prohormone convertase strains, a nematode species, was performed using 0.1% triflu-
1 and 2, propeptidyl-amidating monooxygenase, proteins des- oroacetic acid (TFA) with 50% acetonitrile (CH3CN) on
ignated proSAAS and VGF; after labeling by isotopic tags in symmetry (4.6 mm, i.d. · 250 mm) C18 column for HPLC
extracts of mice with out PC2 and wild type young ones fol- analysis followed by matrix-assisted laser desorption ioniza-
lowing fractionation with RP-HPLC column. Electrospray tion-time-of-flight mass spectrometry (MALDI-TOF). Fur-
ionization mass spectrometric method and tandem-mass spec- thermore, 2–50% acetonitrile and 0.1% formic acid; in the
trometry were used for analysis and identification of above same column; with a flow rate of 200 nL/min is used for
said proteins, respectively. on-line nano-liquid chromatography-quadrupole time-of-flight
An interaction between aquaporin and filaments was tandem-mass spectrometry (nano-LC-Q-TOF-MS/MS) to
reported using liquid chromatography (LC)–tandem-mass confirm the sequence of several naturally occurring peptides
spectrometry method. This interaction was supposed to be as shown in Fig. 3 (Husson et al., 2006). The authors reported
responsible for the lens fiber cell shape (Lindsey Rose et al., that the presence of FMRFamide-like peptide (FLP) and neu-
2006). Andre et al. (2006) reported a LC–ESI-MS/MS and ropeptide like protein (NLP) in wild type strain of C. elegans
MALDI-FTICR method for the identification of tetraspanin, was due to the activity of EGL-3 gene.
which were integral membrane proteins, in a model of human Gallagher et al. (2006) achieved the isolation of glutathione
colon cancer. These identified proteins were integrins, Lu/B- S-transferase isoenzyme (GSTs), for the detoxification of xeno-
CAM, GA733, BAI2, PKC, G, proteages (ADAM10, biotics and endogenous toxicants, using a (150 mm · 4.6 mm)
TADG15) and syntaxins proteins. Rosas-Acosta et al. (2005) Vydac 214TP C4 column with 37% acetonitrile having
reported SUMO-1 and SUMO-3 as stable modified proteins 0.075% trifluoroacetic acid (TFA) in water using HPLC sub-
having half lives more than 20 h by LC–MS. Shin et al. unit analysis of glutathione (GSH) affinity-purified human li-
(2004) described 12 proteins out of 37 different proteins related ver mitochondrial proteins. The authors identified three
with Alzheimer’s disease in the cortex of Tg2576 mice using human liver mitochondrial GST isoenzymes namely hGSTA1
matrix-assisted laser desorption/ionization-time-of flight and hGSTA2 of alpha class GST and hGSTP1 of pi class
(MALDI-TOF) and liquid chromatography–tandem-mass GST subunits. The authors reported three GSH affinity-puri-
spectrometry. The whole phosphoproteome was studied using fied human liver mitochondrial proteins at 14.7, 19.2 and
multidimensional liquid chromatography with electrospray 21.5 min retention times.
mass spectrometric method in eukaryotic living beings Vanrobaeys et al. (2005) analyzed the peptide mixture by
(Metodiev et al., 2004). Soreghan et al. (2003) reported a liquid combination of MALDI MS/MS with off-line liquid chroma-
chromatography and tandem-mass spectrometry method to tography and recognized 377 unique peptides with the identifi-
identify the carbolylated proteins in aged mouse brain homog- cation of 93 proteins. Wang et al. (2005) reported nano-RPLC
enates. Brock et al. (2003) identified K7, K37 and K41 as main as an important method for single and multidimensional pro-
sites of glycation and carboxymethylation of RNase by using tein separation of complex protein mixtures before mass spec-
electrospray ionization liquid chromatography–mass spec- trometric analysis. The authors also reported the effects of
trometry (ESI-LC–MS) method after the incubation of RNase various chromatographic conditions on protein separation
(13.7 mg/mL, 1 mM) with glucose of 0.4 M concentration at such as alkyl chain length in the stationary phase, temperature
37 C for a period of 14 days in phosphate buffer. The average and ion pairing agent including C18 column at 60 C with TFA
value of mass measurement accuracy (MMA) of apomyoglo- instead of heptafluorobutyric acid (HFBA). The influence of
bin was reported by using nano-liquid chromatography-dual alkyl chain length in stationary phase for model protein sepa-
electrospray ionization-Fourier transform-ion cyclotron reso- ration is shown in Fig. 4 at 25 C column temperature using
nance-mass spectrometry (nano-LC-dual ESI-FT-ICR-MS) acetonitrile as mobile phase having 0.1% TFA. Zolla et al.
as 1.09 versus 74.5 ppm (Nepomuceno et al., 2003). (2003) identified the photosystem II (PS II) antenna proteins
62 I. Ali et al.

Figure 1 Analysis of a tryptic digest of a protein band from an Outer membrane protein (Omp) at two various times. (A) In LC
separation, chromatogram represents the high reproducibility of the retention time and peak distribution (B) the measurements of mass of
peptides with doubly protonated at m/z 836.94 at 21.32 and 21.25 min. (C) CID spectra of doubly protonated peptide showed at m/z
836.94 Carlsohn et al. (2006).

Figure 2 Few represented MPC proteins create a large no of MPC peptides Seshi (2006).

on Vydac protein C4 column with 27.5–63.5% acetonitrile, fish from sea and farm by LC-ES/MS/MS study using a nar-
0.05% trifluoroacetic acid in water as mobile phase with row-bore Phenomenex Jupiter C18 column (250 · 2.1 nm) with
1.0 mL/min. flow rate with MS detection in arabidopsis, pea 0.05% (v/v) TFA, 5% (v/v) formic acid in H2O and 0.05% (v/
and tomato. Fig. 5 represents the ion chromatogram of pea v) TFA, 5% (v/v) formic acid in acetonitrile as solvent. Chen
with protein components of PS II. Reh et al. (2006) reported et al. (2005) reported a 2D-LC–MS/MS method to identify
that neither surface area nor pore diameter played an impor- secretory proteins from rat adipose cells. The authors sepa-
tant role in the application of reversed phase for HPLC for rated these proteins using Zorbax 300 SB-C3 reversed phase
proteomics. column (150 mm · 4.6 mm) with flow rate of 700 lL/min of
Monti et al. (2005) identified various proteins by FASTA TFA and acetonitrile. The authors separated 33 protein com-
and protein Prospector software in tryptic peptide mixture of plexes; called as bands; by two-dimensional LC–MS/MS using
Separation of biological proteins by liquid chromatography 63

Figure 3 Comparative study of MALDI-TOF MS spectra (a): C18 HPLC analysis of wild type C. elegans extract and obtained fractions
were further analyzed by MALDI-TOF MS (only fraction 35 is shown in figure). Measured masses were compared with theoretical masses
of FLP and NLP peptides. (b) The analysis of extracts of various C. elegans strains with mutated egl-3 with same procedure as with wild
type strains. Zoom spectrum of fraction 35 of 4 strains namely n588, n150, n729 and gk238 are shown Husson et al. (2006).

a Mono Q HR 5/5 column with sodium chloride from 0.1 M 11 human glycosylphosphadylinositol anchored proteins (GPI-
NaCl in murine erythroleukemic cells. APs) and 35 Arabidopsis thaliana GPI-APs using a 2 cm fused
A nano-HPLC-MS/MS method for the study of low abun- silica Zorbax SB-C18 column with solution A having acetoni-
dance proteins in silico analysis of complex protein samples trile in 1% formic acid/0.6% acetic acid/0.005% heptafluo-
was reported using 5 lm Zorbax SB C18 using buffer A: robutyric acid (HFBA) with 40% B solution containing 90%
95% H2O, 5% acetonitrile, 0.1% formic acid and buffer B: acetonitrile in 1% formic acid/0.6% acetic acid/0.005% HFBA
90% acetonitrile, 10% water, 0.025% trifluoroacetic acid and as mobile phases for half an hour. Hoffert and coworkers
0.1% formic acid (Bihan et al., 2004). Garcia et al. (2005) used (Hoffert et al., 2007) performed LC–MS/MS phosphoproteo-
a nano-flow high performance liquid chromatographic mic analysis of phosphopeptides obtained from membrane
(HPLC) method using 0.1% acetic acid as solvent A and fractions of rat kidney inner medullary collecting duct (IMCD)
70% acetonitrile in 0.1% acetic acid as solvent B with the on a C18 pre-column for desalting the digested peptide mixture
detection by mass spectrometer. Elortza et al. (2006) identified and these peptides were subjected to a Picofrit reverse-phase
64 I. Ali et al.

ble to design a stationary phase that reversibly binds to a

known subset of molecules just by combining affinity chroma-
tography. This kind exploits a well known and defined prop-
erty of analytes which can be used during purification
process. The process can be considered as an entrapment with
the target molecule trapped on a stationary phase while the
other molecules in solution did not trap due to lack of this
property.
Tumor necrosis factor receptor, i.e., factor 6 (TRAF6)
binding proteins, having many heat shock proteins, in osteo-
clast cells were reported by Ryu et al. (2005) using affinity
chromatography followed by mass spectrometric technique.
Matsumoto et al. (2005) studied ubiquitin-conjugated and
ubiquitin-associated proteins in human cells by immunoaffin-
ity chromatography and LC–MS/MS. The authors reported
345 proteins as ubiquitin-related proteome in denaturing
conditions (Urp-D) and 325 proteins as ubiquitin-related
proteome in native conditions (Urp-N). Welch et al. (2005)
studied many potential susceptibility factors, which were oc-
curred in the livers of SJL mice using a C18 pre-column
(100 lm · 2 cm) followed by 5% solvent B (100% acetoni-
trile) for loading of isotope-coded affinity tag (ICAT)-
labeled purified peptide strong cation exchange (SCX) frac-
tions. Furthermore, the authors reported the separation of
Figure 4 Comparative study of model protein separation by these peptides using a (75 lm · 15 cm) self packed Magic
using (A): C4 column (B): C18 at 25 C with an elution order as (1) C18 AQ column with 250 nL/min flow rate of 99.9% H2O
ribonuclease A, (2) cytochrome c (3) bovine serum albumin and (4) in 0.1% HCOOH (solvent A) and 100% acetonitrile (solvent
myoglobin using acetonitrile as mobile phase having 0.1% TFA B). Mass spectrometric analytical study has been done and
with 200 nL/min flow rate Wang et al. (2005). studied the correlation between experimental data with the-
oretical spectra using a SEQUEST program. Senis et al.
analytical column which has the elution of these peptides with (2007) reported liquid chromatography and tandem-mass
0–60% acetonitrile in 0.1% formic acid maintaining 250 nL/ spectrometry, lectin affinity chromatography, biotin/NeutrA-
min flow rate. Fourier transform mass spectrometer having a vidin chromatography for the analysis of transmembrane
nanospray ion source was used to analyze the peptides. The proteins in human platelets and mouse mega-karyocytes.
authors reported CIC-1, LAT4, MCT2, NBC3 and NHE 1 The authors reported unique peptides for 46, 68 and 22 sur-
as solute transporter proteins having new phosphorylation face membrane and intracellular membrane, respectively,
sites. Calvete et al. (2007) determined the compositions of and identified new plasma membrane proteins covering
the venoms of snakes such as Bitis gabonica rhinoceros (West immunoglobulin member G6b-B, a immunoreceptor tyro-
African gaboon viper), Bitis nasicornis (Rhinoceros viper), Bi- sine-based inhibition motif.
tis caudalis (Horned puff adder) using RP-HPLC followed by Immobilized metal affinity chromatography was used for
N-terminal sequencing, MALDI-TOF peptide mass finger- the purification of phosphopeptides from Arabidopsis root cell
printing and CID-MS/MS methods. For this RP-HPLC sepa- culture and reported 79 phosphorylation sites in 22 phospho-
ration, the authors used a Lichrosphere RP100 C18 column proteins having a central role in RNA metabolism using Pep-
(25 cm · 4 mm, i.d.) with 0.1% trifluoroacetic acid (TFA) in Map C18 (300 lm · 5 mm), column and 0.1% TFA with
water as solution A and acetonitrile with different concentra- 20 lL/min. flow rate in a nano-HPLC technique (de la Fuente
tion for different times as solution B. Table 1 presents proteins van Bentem et al., 2006). Cantin et al. (2006) reported up reg-
of total HPLC-analyzed in venom of various snake species. Li ulation of 106 phosphopeptides and 145 phosphorylation sites.
et al. (2007) reported iTRAQ reagents tagging in conjugation Affinity chromatography was reported as an indispensable
with LC–LC MS/MS analytical study advantageous for quan- tool for the separation of complex proteins (Azarkan et al.,
titative study of synaptic proteomes of wild type mice and 2007). Cao and Stults (2000) used immobilized metal affinity
30 UTR-calcium/calmodulin-dependent kinase II a mutant chromatography coupled with electrospray ionization tandem
mice. The authors used 300 lL of buffer having 20% acetoni- MS and Stensballe et al. (2001) described same techniques with
trile, 10 mM KH2PO4 with 2.9 pH to dissolve dried iTRAQ- matrix-assisted laser desorption/ionization (MALDI) MS in
tagged sample and injected into a Polysulfoethyl A column. phosphoproteomic analysis.
The column used was of 150 mm · 100 lm i.d. with a
500 nL/min flow rate of mobile phase. 1.4. Gel permeation high performance liquid chromatography

1.3. Affinity high performance liquid chromatography Basically, Gel Permeation High Performance Liquid Chroma-
tography works on the principle of sizes of the compounds and
Affinity HPLC is a chromatographic method capable to sepa- in this big size molecules eluted first followed by small size
rate biochemical mixtures of highly specific nature. It is possi- molecules. It involves the transport of a liquid mobile phase
Separation of biological proteins by liquid chromatography 65

Figure 5 Identification of protein components of photosystem II by using reversed-phase HPLC-ESI-MS Zolla et al. (2003).

through a column containing a porous material as stationary 1.5. Ligand exchange high performance liquid chromatography
phase. It also called as size-exclusion chromatography and af-
fords a rapid method for the separation of polymeric species. Ligand exchange-HPLC is the advance version of RP-HPLC
Therefore, it is a method of choice for separation of biomole- where the reversed phase column is replaced by ion exchange
cules such as peptides, proteins, enzymes. The stationary phase column. It has been used widely for the analysis of all inor-
is porous solid such as glass or silica, or a cross-linked gel ganic and organic ionic species. In LE-HPLC, anion and cat-
which contains pores of appropriate dimensions to effect the ion exchange columns are used but, nowadays, mixed (anion
separation desired. Tran et al. (2004) reported the separation and cation) columns are also available which improve the sep-
and isolation of proteins from rat liver nuclei by using micro- aration efficiency. In cation exchange chromatography, the
cystin-Sepharose chromatography followed by mass spectrom- stationary phase is usually composed of resins containing sul-
etry. The authors also identified two novel peroxisomal fonic acid groups or carboxylic acid groups of negative charges
proteins, one was peroxisome-specific isoform of Lon protease and, thus, cation metallic species are attracted to the stationary
and the other was made up of an aminoglycoside phospho- phase by electrostatic interactions. In anion exchange chroma-
transferase-domain with an acyl-CoA dehydrogenase domain tography, the stationary phase is a resin, generally, containing
(Kikuchi et al., 2004). primary or quaternary amine functional groups of positive
66 I. Ali et al.

Table 1 Percentage of proteins reported in venoms of various families of snakes by HPLC separation Calvete et al. (2007).
% of total venom proteins
Protein family B. g. rhinoceros B. nasicornis B. g. gabonica B. a. arietans B. caudalis
Bradykinin-potentiating peptides
Dimeric disintegrin 0.3 – 2.8 – –
Long disintegrin 8.5 3.5 3.4 – 2.3
Kunitz-type inhibitors – – – 17.8 –
Cystatin 7.5 – 3.0 4.2 3.2
DC-fragment 5.3 4.2 9.8 1.7 –
svVEGF 0.6 <0.1 0.5 – –
PLA2 – – 1.0 – –
Serine proteinase 4.8 20.1 11.4 4.3 59.8
CRISP 23.9 21.9 26.4 19.5 15.1
C-type lectin 1.2 1.3 2.0 – 1.2
L-amino acid oxidase 14.1 4.2 14.3 13.2 4.9
Zn2+-metalloproteinase 2.2 3.2 1.3 – 1.7
Unknown peptides 30.8 40.9 22.9 38.5 11.5
0.8 0.7 1.2 0.9 0.3

Figure 6 Separated quantity of proteins from C. glutamicum membranes by washing with different solutions Schluesener et al. (2005).

charge and, thus, these stationary phase groups pull solutes of proteins. The neutral buffer (Tris–HCl, pH 8.0) or sodium car-
negative charge. It can be used effectively for the speciation of bonate (pH 11) separated 18% and 26% of total protein from
cationic, anionic and neutral species simultaneously. membranes, respectively, while 6 M urea solution separated
Schluesener et al. (2007) reported anion exchange chroma- 70% and 4 M guanidine thiocyanate separated approximately
tography using an anion exchange column as faster and more 90% of the total protein from the membranes. Metz and
effective technique for the separation and quantification of coworkers (Metz et al., 2006) characterized isolated human
membrane proteins of wild type Corynebacterium glutamicum pancreatic islet proteomes and identified 29,021 peptides
and L-lysine producing strain. They also identified the proteins equivalent with 3365 proteins using two-dimensional liquid
in the membrane of either wild type or the L-lysine. Further- chromatography (2D-LC) followed by ion-trap tandem-mass
more, Schluesener et al. (2005) presented a significant method spectrometric (MS) study. Strong cation exchange (SCX)
for the analysis of membrane proteome of a gram positive bac- fractionations of enzymatic digests of proteins from human
teria, i.e., C. glutamicum using a column (10 cm · 4.6 mm i.d.) pancreatic islet have been carried out on a Polysulfoethyl A
in ion exchange chromatography. Quantities of proteins were (200 · 2.1 mm) column with 10 mM ammonium formate in
separated from C. glutamicum membranes using different water having 25% acetonitrile and 500 mM ammonium for-
washing solutions as given in Fig. 6. The authors reported mate in water having 25% acetonitrile in SCX chromatogra-
2.5 M NaBr as the best washing solution; among various lower phy with a flow rate of 0.2 mL/min. The protein was
concentration solutions of NaBr because it removes 40% of extracted by using urea/CHAPs or TFE.
Separation of biological proteins by liquid chromatography 67

Opiteck et al. (1997, 1998) performed proteomic analysis of and other aliquots by reversed phase C18, (4.6 mm,
fractions of Escherichia coli lysates using combination of i.d. · 150 mm) column (218 TP 5415 Vydac) with linear gradi-
strong cation-exchange (SCX) or size-exclusion chromatogra- ent of acetonitrile and water having 0.1% TFA as mobile
phy (SEC) coupled with RP-HPLC followed by UV and mass phase for two-dimensional separation study in SEC. The
spectrometry detection. Wagner and coworkers (Wagner et al., authors reported that liquid-based isoelectrofocusing-size-
2002) reported a fast multidimensional chromatographic exclusion chromatography (IEF-SEC) was able to separate
method as the combination of first-dimension ion-exchange milligrams of proteins according to isoelectric point and
chromatography with four reversed phase columns for the molecular size. Xiang et al. (2004) reported a liquid chromato-
analysis of small protein and peptides of human haemofiltrate. graphic study of membrane proteins obtained from breast can-
A three-dimensional peptide fractionation approach for the cer MCF7 and BT474 cells using a fused silica strong cation
quantitative proteomic study is reported (Link, 2002) in which exchange (SCX) column of (7.5 cm · 75 lm i.d.) having Poly-
trypsin digested and isotope-coded affinity tag (ICATTM) re- sulforthyl A resin. The authors identified total 313 proteins
agent of a complete proteome lysate is fractionated. Lecchi from MCF7 cell membranes, 602 proteins from BT474 cell
et al. (2003) performed a multidimensional chromatographic membranes and 117 common proteins in MCF7 and BT474
separation using size-exclusion chromatography for the pro- cell membranes as given in Table 2. Fung et al. (2004) stud-
teomic analysis of E. coli (Strain BL 21). The authors used a ied lacrimal-specific praline-rich proteins having significant
TSKG3000SWxL 7 · 300 mm column and KH2PO4 50 mM role in pathogenesis of inflammatory and autoimmune
and NaCl 200 mM in water as mobile phase for some aliquots diseases, in human tear fluid with matrix-assisted laser

Table 2 HPLC identified proteins from BT474 and MCF7 cell membranes Fung et al. (2004).
BT474 % of total MCF7 % of total Common proteins % of total
Protein locations 602 313 117
Mitochondrion 15 2.5 4 1.3 2 1.7
Plasma membrane 49 8.1 24 7.7 3 2.6
Peroxisome 3 0.5 2 0.6 1 0.9
Nucleus 45 7.5 27 8.6 13 11.1
Endoplasmic reticulum 22 3.7 9 2.9 8 6.8
Cytoplasm 35 5.8 27 8.6 13 11.1
Golgi apparatus 2 0.3 1 0.3 0 0.0
Proteasome 4 0.7 1 0.3 1 0.9
Ribosome 22 3.7 22 7.0 18 15.4
Unknowns 392 65.1 195 62.3 58 49.6
Location unclear 15 2.5 1 0.3 0 0.0
604a 100.00 313 100.00 117 100.00
a
2 reported at multiple locations.

Figure 7 A view on 2D-nano-LC–MS/MS analysis. (A) Total ion current chromatogram of 10 SCX fractions received from a C. elegans
extract after 2D-nano LC separation. (B) The ion at m/z 504.34 is selected for fragmentation of 600 mM fraction at 34.5 min. (C)
GSLSNMMRI amide sequence of fragmentation spectra of selected peptides Husson et al. (2005).
68 I. Ali et al.

Table 3 Identification of C. elegans neuropeptides by 2D-nano-LC–MS/MS technique Husson et al. (2005).

Gene Gene similarity Peptides characterized by 2D-nano-LC–MS/MS
FMRFamide-related peptides or FaRPs
LRFamide family
flp-1 C. vulgaris, C. briggsae SADPNFLRFamide
C. redivivus, myosuppressins AAADPNFLRFamide
flp-18 C. briggsae EIPGVLRFamidea
SEVPGVLRFamide
SYFDEKKSVPGVLRFamide
SVPGVLRFamidea
DFDGAMPGVLRFamide
GAMPGVLRFamide

IRFamide family
flp-5 C. briggsae GAKFIRFamide
flp-13 C. briggsae APEASPFIRFamide
AMDSPLIRFamide
ASPSAPLIRFamidea
SPSAVPLIRFamide
SAAAPLIRFamide
AADGAPLIRFamide
MRFamide family
flp-3 TPLGTMRFamide
EAEEPLGTMRFamide
SADDSAPFGTMRFamide
SAEPFGTMRFamide
ASEDALFGTMRFamide
NPENDTPFGTMRFamide
flp-22 SPSAKWMRFamide
flp-6 pQQDSEVEREMM

VRFamide family
flp-9 C. briggsae KPSFVRFamide
flp-11 C. briggsae ASGGMRNALVRFamide
NGAPQPFVRFamidea
SPLDEEDFAPESPLQamide
flp-16 C. briggsae AQTFVRFamide
GQTFVRFamidea
flp-19 C. briggsae WANQVRFamide

Neuropeptide-like protein (NLP) peptides

MSFamide family
nlp-1 C. briggsae, buccalin
drosulfakinin-0 MDANAFRMSFamide
VNLDPNSFRMSFamide
nlp-13 C. briggsae SAPSDFSRDIMSFamide
SSSMYDRDIMSFamidea
SPVDYDRPIMAFamide
nlp-7 C. briggsae LYLKQADFDDPRMFTSSFamidea
(F/M)G(L/F)amide family
nlp-6 C. briggsae, allatostatins A APKQMVFGFamide
GFxGF family
nlp-8 C. briggsae YPYLIFPASPSSGDSRRLV
C. briggsae SFDRMGGTEFGLM
nlp-14 C. briggsae, orcokinin ALNSLDGAGFGFE
nlp-15 C. briggsae AFDSLAGSGFDNGFN
FAFA family
nlp-18 C. briggsae SDEENLDFLE
SPYRTFAFA
SPYRAFAFA
Separation of biological proteins by liquid chromatography 69

Table 3 (continued)
Gene Gene similarity Peptides characterized by 2D-nano-LC–MS/MS
GGARAF-family
nlp-21 C. briggsae pQYTSELEEDE
GGARVFQGFEDE
GGARAFLTEM
nlp-9 C. briggsae TPIAEAQGAPEDVDDRRELE
No multigene family
nlp-11 C. briggsae SPAISPAYQFENAFGLSEALERAamide
nlp-17 C. briggsae GSLSNMMRIamide

Newly characterized peptides

Novel FaRPs
flp-24 C. briggsae VPSAGDMMVRFamide
VPSAGDM(ox)MVRFamide
VPSAGDMM(ox)VRFamide
VPSAGDM(ox)M(ox)VRFamide
flp-26 C. briggsae EFNADDLTLRFamide
FNADDLTLRFamide
GGAGEPLAFSPDMLSLRFamide
Novel NLP peptides
nlp-35 C. briggsae AVVSGYDNIYQVLAPRF
nlp-36 C. briggsae SMVARQIPQTVVADH
nlp-37 C. briggsae NNAEVVNHILKNFGALDRLGDVamide
nlp-38/MIP Insect MIPs, B-type allatostatins TPQNWNKLNSLWamide
SPAQWQRANGLWamide
nlp-39 EVPNFQADNVPEAGGRV
nlp-40 C. briggsae APSAPAGLEEKL
APSAPAGLEEKLR
nlp-41 APGLFELPSRSV
a
Peptides, which have Mowse scores below the threshold required for identity.

desorption/ionization-time-of-flight mass spectrometry fol- mechanisms work simultaneously in CEC and several combi-
lowed by size-exclusion high performance liquid chromatogra- nations are possible.
phy. The authors recognized some lacrimal-specific proteins. The separation and identification of some proteins was per-
The success of C. elegans (a nematode), genome project gave formed in foam cells with capillary liquid chromatography fol-
a typical knowledge of neuropeptide signaling. Neuropeptide lowed by mass spectrometry (Yang et al., 2007). Reinhardt and
are originated from proprotein peptide precursor genes. Lippolis (2006) reported that out of 120 proteins, only 15 in
Husson et al. (2005) performed a peptidomic analysis of cow milk fat globule membranes (MFGM), had similarity with
C. elegans using a strong cation exchange column (Bio-SCX, previously studied mouse or human MFGM proteome using a
500 lm · 15 mm) attached with a C18 pre-column and 2% ace- micro-capillary liquid chromatograph which was linked with a
tonitrile (ACN), 0.1% formic acid (FA) with water taking a nanospray-tandem-mass spectrometer. Casado et al. (2005)
flow rate of 30 lL/min. in a two-dimensional nano-scale liquid identified 111 human nasal mucous proteins in nasal lavage
chromatography-quadrupole time-of-flight tandem-mass spec- fluids (NLFs) of ten volunteers (patients) using a capillary li-
trometry (2D-nano-LC-Q-TOFMS/MS) method and reported quid chromatography-electrospray quadrupole-time-of-flight
a total ion current (TIC) chromatogram for every nano- mass spectrometric method. Lominadze et al. (2005) analyzed
LC–MS study shown in Fig. 7. The authors arranged 60 human neutrophil granules responsible for chemotaxis, phago-
neuropeptides given in Table 3 and reported their similarity cytosis and bacterial killing susing two-dimensional micro-
with neuropeptides of vertebrates or invertebrates. capillary chromatography, reversed phase micro-capillary
liquid chromatography followed by electrospray ionization
1.6. Capillary high performance liquid chromatography tandem-mass spectrometry (2D HPLC ESI-MS/MS) technique
and reported 286 proteins. Yuan and coworkers (Yuan and
A hybrid technique of HPLC and CE was developed in 1990 Desiderio, 2005) studied low molecular mass peptides in
and is called as Capillary Electro-chromatography (CEC). It human cerebrospinal fluid (CSF), i.e., amyloid-like protein 1,
is expected to combine high peak efficiency which is character- secretogranin I, granin like neuroendocrine peptide precursor
istic of electrically driven separations with high separation and neurosecretory protein using capillary liquid chromatogra-
selectivity. CEC experiments can be carried out on wall coated phy followed by quadrupole time-of-flight mass spectrometry.
open tubular capillaries or capillaries packed with particulate Boisvert et al. (2003) identified 200 novel arginine-methylated
or monolithic silica or other inorganic materials as well as or- proteins using micro-capillary liquid chromatography with
ganic polymers. The chromatographic and electrophoretic electrospray ionization tandem-mass spectrometry. Capillary
70 I. Ali et al.

chromatography separation method was reported as a best of 5.0 lm Jupiter C18-bonded particles. The mobile phases
separation method in combination with mass spectrometry were 0.2% acetic acid and 0.05% TFA in water (Solvent sys-
for complex protein mixtures due to high sensitivity of this tem A) and 0.1% TFA in 90% acetonitrile in water (Solvent
method (Shen and Smith, 2002). Zhang et al. (2003) identified system B) was achieved by MS/MS. Meek et al. (2004) re-
145 unique peptides mapping 57 unique human serum proteins ported a descriptive proteomic analysis of interphase and mito-
using micro-capillary liquid chromatography electrospray ion- tic 14-3-3-binding proteins using 14-3-3 zeta affinity column
ization MS/MS method. The nano-LC-FTICR analysis of and many new 14-3-3 binding proteins were recognized by mi-
0.5 pg of a bacterium Deinococcus radiodurans proteome cro-capillary high performance liquid chromatography tan-
was carried out using a 14.9 lm inner diameter separation cap- dem-mass spectrometry. These proteins had a significant role
illary that was packed with 3 lm diameter stationary phase in cell cycle regulation, metabolism, protein synthesis, protein
particles (Shen et al., 2004). Martinovic et al. (2000) reported folding, proteolysis, nucleic acid binding etc.
that capillary isoelectric focusing (CIEF) in combination with
FTICR-MS improved 10 throughputs for detection of pro-
teins. Zhang and coworkers (Zhang et al., 2007) reported 1.7. Comparison of various chromatographic methods
CEC of enriched peptides, i.e., nitrotyrosine-containing pep-
tides in complex proteome sample of mouse brain homoge- Among various chromatographic methods used in proteomic
nate. The mobile phases used were 0.2% acetic acid, and analyses the order of application is reversed phase > gel per-
0.05% TFA in water as solvent A and 0.1% TFA in 90% ace- meation > ligand exchange > affinity. During our search of
tonitrile as solvent B. Metz et al. (2006) used reversed-phase literature it was found that the maximum papers on proteomic
capillary liquid chromatography for separation of vacuum analyses were on reversed phase high performance liquid chro-
dried peptide fractions using reversed-phase capillary column matography. It is due the fact that this kind of chromatogra-
(65 cm · 150 lm) of fused silica capillary; packed with slurry phy is well developed. There are many types of reversed

Table 4 A comparison of proteomic analyses on various chromatographic techniques.

Proteomes Columns Mobile phases References
Reversed phase high performance liquid chromatography
Complex protein mixture C18 column Acetonitrile–TFA Wang et al. (2005)
Cycloskeletal proteins and enzymes PepMap C18 (250 mm · 0.3 mm) – Babusiak et al. (2007)
Peptides of Caenorhabditis elegans C18 (250 mm · 4.6 mm i.d.) Acetonitrile–formic acid Husson et al. (2006)
S-transferase isoenzyme Vydac 214TP C4 (150 mm · 4.6 mm) Acetonitrile and water with TFA Gallagher et al. (2006)
Photosystem II antenna protein Vydac C4 Acetonitrile–water–TFA Zolla et al. (2003)
Proteins of rat adipose cells Zorbax 300 SB-C3 (150 · 4.6 mm) Acetonitrile–TFA Chen et al. (2005)
Complex protein mixture Zorbax SB-C18 Buffer–acetonitrile–FA Bihan et al. (2004)
Buffer–acetonitrile–TFA
GPI-APs protein Zorbax SB-C18 Acetonitrile with acids HFBA Elortza et al. (2006)
(different combinations)
Phosphopeptides of rat kidney Picofrit RP column Acetonitrile–FA Hoffert et al. (2007)
IMCD
Venoms of various snakes Lichrosphere RP100 C18 Water–acetonitrile–TFA Calvete et al. (2007)
(250 · 4 mm with 5 lm)
Affinity high performance liquid chromatography
Peptides of liver of mice Magic C18 AQ (75 lm · 15 cm) Water–acetonitrile–FA Welch et al. (2005)
Phosphopeptides of Arabidopsis PepMap C18 (300 lm · 5 mm) – de la Fuente van
Bentem et al. (2006)
Ligand exchange high performance liquid chromatography
Proteomic analysis of E. coli 218 TP 5415 Vydac C18 RP column Acetonitrile–water–TFA Lecchi et al. (2003)
strain BL 21 (150 · 4.6 mm)
Pancreatic islet Proteome Polysulfoethyl A column (200 · 2.1 mm) 10 mM Ammonium formate Metz et al. (2006)
buffer–water and acetonitrile
Membrane proteins of breast Polysulfoethyl A resin (7.5 cm · 75 lm i.d.) – Xiang et al. (2004)
cancer MCF7 and BT474 cells
Peptidomic analysis of Bio-SCX column (15 mm · 500 lm) Water–acetonitrile–FA Husson et al. (2005)
Caenorhabditis elegans
Capillary electro-chromatography
Protein of Helicobacter pylori ReproSil-Pur C18-AQ (17 cm · 50 lm i.d.) – Carlsohn et al. (2006)
Tryptic peptide mixture of fish Phenomenex Jupiter C18 (250 · 2.1 nm) Acetonitrile–TFA–formic acid Monti et al. (2005)
Synaptic proteomes of wild Polysulfoethyl A (150 mm · 100 lm i.d.) Buffers–acetonitrile–10 mM Li et al. (2007)
type mice KH2PO4
Proteomic analysis of E. coli TSKG3000SWxL (300 · 7 mm) Water–50 mM KH2PO4–200 nM Lecchi et al. (2003)
strain BL 21 NaCl
Vacuum dried peptides Jupiter C18 RP capillary column Water with TFA and FA, water Metz et al. (2006)
(65 cm · 150) and acetonitrile with TFA
Separation of biological proteins by liquid chromatography 71

phase stationary phases available, which can be used for anal- trometer detectors this technique has achieved heights in
yses of proteomes. Besides, the reversed phase columns are analysis work. It can detect molecules at the level of the
capable to work with a wide range of mobile phases, enhancing nanomole. Hence, it is useful in proteomics and genome re-
the application range of reversed phase chromatography. On search. Many kinds of liquid chromatography such as re-
the other hand, gel permeation HPLC is also useful for proteo- versed phase high performance liquid chromatography,
mic separation and identification due to a wide variation in the affinity high performance liquid chromatography, gel perme-
sizes of proteins. Ligand exchange is also useful as proteins ation high performance liquid chromatography, ligand ex-
have charges, which may be exploited in this kind of chroma- change high performance liquid chromatography and
tography. Affinity and capillary electro-chromatographic tech- capillary high performance liquid chromatography have been
niques have also been used in proteomic area. Later technique used in proteomic research. More advance paraphernalia is
is more useful as it needs little amount of sample and also has required to achieve the detection at picomolar and femtomo-
low detection limit. Therefore, all these techniques are impor- lar levels, which are required in proteomics and genome re-
tant and useful for proteomics analyses depending on the type search. Besides, the mechanism and medication of various
and nature of the proteins to be analyzed. They have their own diseases can be understood by using the concept of chirality
merits and demerits, which cannot be discussed in detail here. in proteomic. Chiral chromatography may be a useful tool
However, the comparative features can be seen from Table 4 for the proteomic interactions.
having applications of different kinds of chromatographic
methods.
Acknowledgements
1.8. Chirality and chirality and protomics
Authors are thankful to Uttarakhand State Council for Sci-
ence and Technology, (UCOST) Dehradun, India for provid-
It is well known-fact that millions of our bodies proteins inter- ing financial assistance to complete this work.
act among themselves and with the biological environment,
i.e., with cell, tissue, organelle, protoplasm and other cellular
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