Musculoskeletal ProteinAnalysis Techniques -
Srinath Kamineni1*, Monica Manepally1, Ellora P Kamineni1
1Elbow Shoulder Research Centre, Department of Orthopedic and Sports Medicine, University of Kentucky, Lexington, KY, USA
Srinath Kamineni Elbow Shoulder Research Centre, University of Kentucky, USA.E-mail:
Received: August 18, 2016; Accepted: September 12, 2016; Published: October 26, 2016
Citation: Kamineni S, Manepally M, Kamineni EP (2016) Musculoskeletal ProteinAnalysis Techniques - A Review. J Rheumatol Arthritic Dis 1(2): 1-9.
There have been a large number of experimental methods
for purifying and analyzing proteins from the sample of interest.
Determination of protein concentration is often the key step and is
common to many applications in protein research and life sciences.
The most commonly used quantitative techniques to measure
the protein concentration are Amino acid analysis, Biuret assay,
Bradford assays, Folin- Lowry assay, Bicinchoninic (BCA) assays, UV
absorbance assays and Antibody-based assays such as ELISA and
Western Blotting. Some of the qualitative methods that are used to
detect different types of proteins and amino acids are Ninhydrin test,
Xanthoproteic test, Millon’s test, Sulfur test, Hopkins-Cole test, and
Nitroprusside test. Chromatographic analysis can also be carried
out on an either qualitative or quantitative basis to purify complex
protein mixtures based on their properties such as size, solubility,
charge, hydrophobicity and bio-specific interaction. Selection
of these assays is based upon the ease of performance, range of
concentrations, sensitivity, and interfering substances contained in
the complex mixture. The purpose of this review is to provide an
assessment of commonly used chromatographic and colorimetric
methods for the determination of protein concentration.
Proteins are macromolecules which are made up of along
chain of amino acids. There are about 20 types of amino acids and
each vary in size, polarity, shape, hydrophobicity and chemical
reactivity and thus every single protein has different molecular
structure with its own particular sequence and number of amino
acids. The determination of protein concentration in a solution
of interests an important aspect of analysis and has many
applications in various clinical and research laboratories. The final
success of research depends on choosing an appropriate essay by
considering various factors, including the presence or absence
of non-protein agents, the quantity of protein present in the
sample and its composition which includes the content of amino
acids. There are wide ranges of assays used to quantitatively and
qualitatively assess the concentration of protein present in the
sample. This article provides the review of the different methods
of protein analysis, in current use.
Methods of Protein estimation
Quantitative protein analysis is an important tool in scientific
research and development labs in order to understand the
dynamics of a cell or other biological samples. The necessity for
assessing the levels of particular proteins in samples is growing
rapidly especially in the medical field to provide more specific
diagnostic information. Some of the commonly used quantitative
assays are UV absorbance assays, Colorimetric assays such as
Biuret, Bradford, Folin-Lowry and Bicinchoninic (BCA) assays
and Antibody-based assays such as ELISA and Western Blotting.
Protein determination using Absorbance at 280 Nm
Spectrophotometry is one of the most extensively used
protein quantization assay that uses UV- Visible spectroscopy to
access the concentration of protein in the sample. This method is
advantageous dueto its simplicity, specificity, speed and low cost.
Near UV Absorbance (280 Nm)
A large number of bioanalytes can be detected by the
absorption of ultraviolet radiation. Protein, including that
in tissues, absorbs ultraviolet light quite strongly due to the
presence of aromatic rings in the side chains of amino acids,
tryptophan, tyrosine and phenylalanine. The absorbance of UV
light is higher in tryptophan and tyrosine than phenylalanine and
tryptophan is the only amino acid that absorbs UV at a maximum
wave length between 275and 280 nm. But an absorbance of
260 nm is shown when the phenylalanine and disulfide bonds
are formed between two cysteineresidues. UV absorption
spectroscopy is relatively sensitive when compared to other
methods and can measure protein concentrations as low as 10
μgcm3.It is a non-destructive technique as the protein sample
can be recovered and reused further which is considered as one
of the advantages. But the main disadvantage is the presence of
chromospheres like nucleic acids that also absorbs UV light at
280 nm and affects the measurement of protein concentration,
but this difficulty is overcome by quantification (measurement of
absorbance) at two different wavelengths. From this technique,
protein concentration is easily calculated by knowing the molar
absorptive and measured absorbance of the sample solution
at 280 nm. Also a calibration curve that is plotted from several
known amounts of protein can be used for comparison in order
to assess the unknown protein concentration.
Far UV Absorbance
The concentration of the proteins that lack aromatic amino
acids and disulfide linkage can be assessed using “far UV
absorption” as they do not absorb near UV radiation. The major
chromospheres in this spectral region are the peptide bond with a
wavelength ranging from 190 nm to 220 nm, whereas in the near-
UV spectral region aromatic residues and disulfide bonds are the
main chromospheres. But at 190 nm, oxygen strongly absorbs UV
light, thereby reducing the light available for measurement and
also the output of deuterium arc sources is low at this particular
wavelength. Hence it is convenient to measure the absorption
at a wavelength of 205 nm in far UV region and the protein
concentration can be measured to less than 1 μg cm –3. The
only difficulty while working at these lower wavelengths is the
absorbance of UV light by the buffer and additional components
One of the simplest and the most common assays the Biuret
Protein assay. The main principle of this assay is under alkaline
conditions, cupric ion complexes with the adjacent peptide
bonds in protein and forms an intense purple color complex.
This reaction arises only with the presence of peptide bonds and
not with amino acid side chains. The maximum intensity of the
color is attained after fifteen minutes and the formed complex
remains stable for several hours. The intensity of the color is
directly proportional to the protein concentration, which is
measured spectrophotometrically at 540 nm. This method
requires a large amount of sample as it measures those protein
concentrations ranging from 0.5 toapproximately5 mg/ml. Very
few materials (e.g. Tris and amino acid buffers) interfere with
this assay, which is considered as one of its advantages and the
only disadvantage is its low sensitivity .
Zaia et al. tested biuret, Lowry/ Hartree, P-chloranil, UV-
280 nm, and UV-260/280 nm for the estimation of total proteins
in seven rat tissues adrenal, spleen, whole brain, small intestine,
pancreas, liver and epididymal fat pad. The sensitivity of Lowry/
Hartree reaction was high for the proteins and comparatively
low for Biuret reaction. However, a good correlation was noticed
between the results of these two assays. As Lowry/hartree
assay was more of time-consuming, the authors suggested the
biuret method for five tissues adrenal, spleen, whole brain, liver,
small intestine and UV-260/280 nm for whole brain, liver, and
pancreas and p-chloranil assay for epididymal fat pad. In another
study by Jenzano et al., the accuracy of biuret, Lowry, BCA,
CB assay in either phosphoric acid formulation or in HCI
formulation proposed by Sedmak and Sidney , were evaluated
for the assay of protein concentration in human mixed saliva.
The data of amino acid analysis results were compared with
the values of the colorimetric assays. With BSA as the standard,
the concentrations determined by the Biuret assay were more
accurate to those obtained by AAA. Due to the presence of very
few aromatic amino acids low values were obtained with the
Lowry method. Dawnay et al.  have surveyed 5 methods and
concluded biuret assay as a simple, rapid and suitable method
for the assay of serum proteins. Sapan et al.  recommended
using Biuret assay, quantitative amino acid analysis and micro-
Kjeldahl technique, when the amount of unknown protein in
the sample does not match the standard. Since the early 1980s,
biuret was the most commonly employed technique, but its only
disadvantage is the lack of sensitivity for detecting the proteins
which are in small volume of samples.
Bradford (Bio-Rad) Method
Bradford assay is a dye-based assay used to measure
the protein content in cell fractions. It measures protein
concentrations ranging from 10and 100 μg/ml. This assay relies
on the change in the spectral shift from 465 nm to 595 nm, due to
the strong binding of negatively charged Coomassie Brilliant Blue
(CBB)G-250 dye to hydrophilic arginine and hydrophobic amino
acid residues in the protein sample under acidic conditions that
result in the formation of a blue color complex. The intensity of
the color complex depends upon the concentration of the protein
which can be measured at 595 nm. This colorimetric assay is
more rapid and compatible with most of the buffers, salts and
solvent sand less prone to interference by various compounds
that are present in protein samples .
In one of the studies, Dilena et al. compared the linearity,
precision, comparative bias, and practicability of the coomassie
brilliant blue technique of Heick et al. , biuret technique ,
trichloroacetic acid turbidimetry , and Ponceau-S dye binding
, for urinary protein determination and the results reported
low precision and linear range for CBB assay. And comparatively
biuret technique had better precision and linear range, but
required an undesirably large volume of sample. In another study
by Swain et al., the accuracy of two dye-binding procedures,
Cellulose-Dye (CD) and the Coomassie Brilliant Blue (CBB) were
compared with a Gel Filtration/modified Biuret method (GFB)
for measurement of total protein in the urine. Indistinguishable
results were obtained for the three assays for the urine sample
containing high protein concentrations, whereas lower values
were obtained for the two dye binding assays than the GFB
method when normal protein samples were analyzed.
Hence, although the standard Bradford technique was very
sensitive and accurate, there were difficulties associated with
the identification of immunoglobulin light chain urinary proteins
and cerebrospinal fluid proteins for clinical laboratory analysis.
Macart et al. approached this problem by adding Sodium
Dodecyl Sulfate (SDS) to the sample prior to the addition of
Comassie Brilliant Blue (CBB) reagent and concluded that the
modified method provides equal reactivity to human albumin, IgG
and transferring. Wimsatt et al. applied the latter to urinary
proteins and also determined it as a simple and appropriate
method. Perini et al. compared the CBB (Bradford) method
and the CBB-SDS method for urinary protein determination and
recommended using the CBB-SDS assay.
On the other hand, the original Bradford method was also not
suitable for the estimation of proteins in collagen rich samples.
Duhamel et al. found out that inclusion of SDS in the dye
binding assay induces a 4-fold increase in the color response
of three collagen proteins (Col I, III and IV) and decrease in the
color yield for other non-collagen proteins. Lopez et al. also
concluded that this modification of Bradford assay minimizes
the large variations of response between collagen and noncollagen
proteins. In a Bradford-micro assay procedure, Pande et
al. have reported that co-precipitating proteins with calcium
phosphate in an ethanolic medium before the addition of dye
reagent eliminates interfering substances and detergents.
Bicinchoninc Acid (BCA) Assay
BCA assay is a copper-based colorimetric assay which depends
on biuret reaction where the proteins back bone chelatescupric
ions and reduces them to cuprous ions under alkaline conditions.
When the seions react with the organic dye, bicinchoninic acid, an
intense purple-colored product is formed which can be measured
spectrophotometricallyat562 nm, against a standard curve of
absorbance from varying Bovine Serum Albumin (BSA). The
incubation temperature determines the rate of color formation
in BCA assay and its unique advantage over the other assays is its
compatibility with samples containing up to 5% detergents and
denaturants. But, due to its sensitivity to the presence of reducing
sugars in the buffer that interfere with the dye, few reducing
agent-compatible dyes are available. It can measure protein
concentrations rangingfrom0.5 μg/mL to 1.5 mg/mL .
The Amino Acid Analysis (AAA) research group Alterman et
al. compared the quantization of range of proteins by AAA
with the BCA, Bradford and Lowry assays. The reports indicated
that only the values obtained with the BCA assay were consistent
with AAA than both the BCA and Lowry assays. Keller and
Nellville have examined the BCA, Biuret, Bio-Rad Coomassie
Blue and Pierce BCA methods for the assay of total protein in
human milk. Though there was a good correlation between the
values obtained from the colorimetric assays with the standard
micro-Kjehdahl method, only BCA assay displayed the greatest
accuracy and consistency. In another study, Fountoulakis et
al. compared the BCA, Lowry and CB methods to analyze the
glycosylated and non-glycosylated proteins. For the glycosylated
proteins, the values obtained with those three colorimetric assays
have not displayed significant correlation with the standard
amino acid analysis, but for the non-glycosylated proteins, the
concentrations determined by BCA assay only were accurate.
Lowry assay is based on both the biuret reaction and Folin-
Ciocalteau reaction and is considered as one of the most accurate
method in determining protein concentration. It can measure
protein concentrations ranging from0.01–1.0mg/mL in a sample.
By mixing the protein sample with copper sulphate solution and
Folin reagent (a mixture of sodium Tung state, molybdate and
phosphate) under alkaline conditions, peptide bonds react with
copper and produces cuprous ions. The reaction further involves
the reduction of Folin reagent and oxidation of aromatic amino
acid residues, tyrosine and tryptophan. The end product of this
reaction is the formation of blue-purple color which can be
quantified by reading the absorbance at a wavelength of 750 nm
against a standard calibration curve of Bovine Serum Albumin
protein solution. This assay is more susceptible to interference
by arrange of substances, including buffers, drugs, nucleic acids,
and sugars. As the amount of the colored product depends upon
the content of tryptophan and tyrosine residues in the protein,
higher absorbance values are observed in those proteins that
comprise large mole fractions of amino acid residues. In order
to estimate the absolute value for protein concentration in this
assay, the protein being assayed should also be used to construct
the calibration curve . Despite its recognition in various
studies, there were also many problems as cited above [5, 8]
with the traditional Lowry assay, such as susceptibility to various
interfering substances, reagent instability and also requires
high time to develop color. However, with the advance of more
accurate and sensitive assays, the use of Lowry assay has reduced
in recent years.
Amino acids react with reagents and can be identified in a
sample solution as they exhibit typical color reactions due to the
presence of specific characteristics defined by their unique side
chains for each of them and also it is the side chain that dictates
the chemical properties of amino acids. The most commonly used
qualitative methods to detect different types of proteins and
amino acids are described below.
Ninhydrin is a strong oxidizing agent used in amino acid
analysis for the precise determination of protein quantities. It
is mainly used as a detector in liquid chromatography methods
coupled with ninhydrin post-column derivatization systems.
The reagent which is initially yellow reacts with free alpha
amino groups present in all amino acids, proteins, or peptides
and forms a deep blue or purple colored complex known as
Ruhemann’s purple. Its absorb an ceis measured at 570 nm using
The Xanthoproteic test uses a nitration reaction to determine
the presence of proteins in a solution. When the sample is treated
with a hot, concentrated nitric acid it reacts with aromatic amino
acids such as phenylalanine, tyrosine and tryptophan and forms
a yellow colored product known as Xantho protein. With the
addition of strong base such as NH3 or NaOH, it further changes
to deep-orange color. So this test gives a positive result in those
proteins which contain amino acids that have aromatic rings in
their side chains.
Millons reagent is the solution containing mercuric nitrate
that is dissolved in nitric acid and is used to identify the presence
of phenolicamino acids such as tyrosine and its derivatives. By
mixing the sample with this reagent and heating it gently results
in the formation of reddish-brown color precipitate signaling the
presence of tyrosine. Some of the proteins containing aphenolic
hydroxyl group initially form a white precipitate and changes to
red when heated which is also considered as a positive result.
Sulfur-containing amino acids such as cystine, cystine and
methionine respond to this test. By boiling the sample with NaOH,
organic sulfur in these amino acids partially convert to inorganic
sulfide and produce sodium sulfide. This reaction can be further
detected by combining the sulfide solution with lead acetate. The
formation of black or gray precipitate of PbS (lead(II)sulfide)
is considered as a positive test for sulfur-containing amino
This chemical test is used to identify the presence of
tryptophan, the only amino acid containing in dole group. When
the protein solution is mixed with Hopkins- cole reagent the
in dole ring reacts with the glyoxylic acid in the presence of
concentrated sulfuric acid and forms a violet or purple colored
product, signifying a positive result.
This test is used to detect the presence of sulfur containing
amino acid, cysteine in a protein. When the sulfhydorxyl group
reacts with sodium nitroprus side in alkaline solution, it yields a
red colored product indicating a positive result of the presence of
amino acid cysteine.
Amino Acid Analysis
Amino acid analysis is an appropriate tool for accurate
determination of protein concentrations. It provides complete
information about the relative amino acid composition, including
free amino acids that are often well enough to identify of proteins.
The analysis procedure involves protein hydrolysis in the first
step and separation, detection and quantification by HPLC
analysis in the second step. Acid hydrolysis, which can be used
in both liquid and gas phase mode is the most frequent method
used to hydrolyze a protein sample into its individual amino
acid components. The sample can be dried in a vacuum after
hydrolysis and is dissolved in loading buffer. But this technique
can have varying effects on sensitive amino acids like tryptophan,
serine, threonine, methionine, cysteine.To reduce losses
during hydrolysis, several investigations were done to use
different protective agents such as phenol, in dole, thioglycolic
acid and alternative non-oxidizing agents such as 4M methanesulfonic
acid. Also, the introduction of microwave radiation
energy for hydrolysis of protein samples allows the hydrolysis
time reduction from many hours to few minutes.
In the second step of analysis, the liberated amino acids are
separated by ion-exchange chromatography. It is coupled with
ninhydrin or o-phthalaldehyde post-column derivatization
system which can measure from5 μgto 10 μg of protein sample
per analysis and can be used with samples comprising small
amounts of sodium or lithium buffer components. Also Reversed-
Phase High Performance Liquid Chromatographic (RP-HPLC)
technique can be used for separation of amino-acids with online
pre-column Ortho-Phthalaldehyde (OPA) derivatisation
and fluorescence detection providing higher sensitivity to the
system. Amino acid analysis, as cited above [8, 22, 24], still
remains the “golden standard technique” for absolute protein
and peptide quantification in various scientific studies.
ELISA is the most sensitive and simple biochemical technique
used for qualitative and quantitative determination of proteins
secreted from cells. There are two main types of ELISA that can
detect and quantify antigen or anti body concentrations: Direct
ELISA and indirect ELISA. The indirect sandwich assay is a best
choice to detect a protein with multiple epitopes. It is a sensitive
assay which measures the antigen concentration between two
layers of antibodies. In this rapid test, an unknown amount of
antigen is immobilized onto a polystyrene surface which acts
as a solid phase and then a specific antibody which is diluted
in blocking buffer is coated over the surface that binds to the
antigen. An enzyme linked antibody molecule is also added
which is allowed to bind to the immobilized antigen. In the final
step a chromogenic substrate for the enzyme is added to the
antigen/ antibody/enzyme complex and causes color to develop.
The intensity of color developed is directly proportional to the
concentration of bound antibodies and can be measured with a
spectrophotometer . The stages of direct ELISA are similar
to indirect ELISA, but in direct format the enzyme conjugated
anti body is directly added and a substrate for this enzyme is
added which changes color upon detection of the enzyme .
Topping et al.  devised an enzyme-linked immunosorbent
assay to measure the urinary Retinol-Binding Protein (RBP) for
the detection of Tubular Proteinuria. In another study, Lucertini
et al.  detected RBP proteins in human serum and urine
by using a double-antibody “sandwich”-type enzyme-linked
immunosorbent assay. In both the studies, the results correlated
well when compared with the latex immunoassay, suggesting
ELISA as a simple, sensitive and an alternative method for the
measurement of tubular function.
The western blotting technique which is also known as
immune blotting, is a powerful tool used for the qualitative and
semi-quantitative determination of a particular protein in a
complex mixture. In principle, the method allows the separation
of proteins according to their size via gel electrophoresis and
identification of the target protein using specific antibodies. After
the protein separations they are electrophoretically transferred
from SDS-gel onto nitrocellulose or Polyvinylidene Fluoride
(PVDF) membrane and probed with approximately matched anti
bodies that detect the target protein. Then a secondary anti body
conjugated to a reporter enzyme or fluorescent dye is added to
bind to the primary anti body and produces a color proportional
to the concentration of protein. The most sensitive detection
methods use secondary anti bodies which are linked to two
enzymes Alkaline Phosphates(AP) and Horseradish Peroxidase
(HRP) that produce a colored precipitate on the membrane for
colorimetric or fluorometric detection. Hossenlopp et al. 
used the western blotting technique to detect Insulin-like Growth
Factor (IGF) binding proteins in normal and hypo pituitary
human serum and the isolated binding proteins were further
measured by titration assay.
Detection and estimation of proteins Ingles
Two Dimensional Gel Electrophoresis
2-DE is one of the widely used methods to classify a
large number of proteins in complex mixtures based on two
independent properties in two dimensions in 2D gels. In the first
dimension, proteins are separated based on an isoelectric point
(pI) and this method is known as isoelectric focusing. When the
sample is placed on the gel with a pH gradient, protein based on
their charges migrate along the pH gradient in the presence of an
electric field. They move up to a certain point in the gel where
their pI equals to the pH. Immobilized pH Gradient (IPG) gel strips
can be used toper form IEF technique which can later be used in
second dimension separation technique, SDS-PAGE, where the
proteins are classified based on their Molecular Weights (MW).
Sodium Dodecyl Sulfate (SDS), which is an anionic detergent,
is typically added to coat the proteins with a uniform negative
charge. As it binds uniformly to all the proteins, they have similar
charge-to-mass ratios and similar shapes. Now based on their
molecular weight SDS- treated proteins will migrate towards the
anode when placed in an electric field. SDS-PAGE is highly used
to test the purity of proteins during chromatographic or other
To visualize the separated proteins in the gel, the most
commonly used staining methods are silver staining, Coomassie
brilliant blue dye stain and fluorescent staining. In silver
staining, the silver colloid is deposited onto the surface of the gel
which binds to certain functional groups of proteins. On exposure
to ultra-violet light, the silver gets darkened. The amount of silver
can be related to the darkness, and therefore the concentration
of protein at a given position on the gel. The separated proteins
after staining can be recognized using Liquid Chromatography-
Mass Spectrometry (LC-MS). The protein band that is cut from
the gel issolubilized and digested into peptide fragments using
a protease enzyme which is mostly trypsin. The short peptide
fragments are then isolated by liquid chromatography and
subjected to MS/MS analysis to further sequence them.
MS is an analytical tool used to detect and identify and provide
quantitative information of the proteins in a manner conceptually
similar to Edman degradation. “Tandem Mass Spectrometry (MS/
MS)”analysis consists of two stages, where in the first approach,
fragments are ionized by the ion source electro spray ionization
(ESI) or Matrix Assisted Laser Desorption Ionization (MALDI).
Then the mass analyzer separates the charged, gas-phase ions
using an electrical and/or magnetic field and selects the peptide
ions based on their mass and charge (MS1). In the second stage,
the precursor ions are striked with neutral gas molecules in the
collision cell of the mass spectrometer. They are fragmented into
smaller pieces and separated again based on their m/z ratios
and intensities (M/S 2). The fragmentation pattern programmed
by the tandem spectrum allows the identification of peptide
sequence and compared with a predicted MS spectrum that can
be found in the protein sequence databases such as IPI, SEQUEST,
Mascot and Swis-Prot . Most of the chromatographic
techniques are coupled with mass spectrometry due to the high
sensitivity and specificity of its detectors .
Manabe et al. used the 2-DE technique in the absence
of denaturing agents to separate and analyze the native human
plasma proteins, in which isoelectric focusing was used in the
first dimension, followed by electrophoresis in a 4-21% linear
gradient slab gel in the second dimension. In another study, Dale
et al.  have described a technique to separate blood serum
proteins by employing isoelectric focusing in an acrylamide gel
in the first dimension followed by electrophoresis in the same
medium. The authors have also suggested using this technique
on CSF and urinary proteins. Chatterji et al. applied 2-DE
technique to extract the serum proteins from a tumor bearing
mice and detected around 46 proteins by analyzing them using
MALDI-TOF/TOF. Li et al. compared five proteomic methods,
2-DE-MALDI-TOF-MS/MS, 2-D HPLC followed by tryptic digestion
of each fraction and micro capillary RP-ESI-MS/MS , On-line and
Off-line 2-D HPLC with micro ESI-MS/MS, 2-D HPLC followed by
optimizing the fractions with nano RP-HPLC-nano ESI-MS/MS to
characterize the human serum proteins. Their results reported
the identification of 37 proteins by all five approaches, but only
2-DE method provided more data on pI-altered isoforms of the
proteins in the serum and also estimated the relative abundance
of identified proteins.
Chromatography refers to a set of techniques used to purify
complex protein mixtures based on their properties such as size,
solubility, charge, hydrophobicity and bio-specific interaction.
Chromatographic analysis can be carried out on an either
qualitative or quantitative basis. Qualitative analysis confirms
the presence of a specific protein in a test sample by relating the
retention time of the peaks in the chromatograph with that of
a reference sample of the test protein obtained under identical
chromatographic conditions. Quantification of a given analytics
also based on the construction of a calibration curve. The area of
each peak in a chromatogram is obtained by the product of the
Height of the Peak (HP) and Width at half the Height (WH) and
is proportional to the amount of the analyte producing the peak.
As this procedure is time consuming, these can be programmed
to compute retention time and peak area and relate them to
High-Performance liquid Chromatography (HPCL)
HPLC is a form of column chromatography used to analyze
proteins based on size, charge or over all hydrophobicity.
The sample is dissolved in a small volume of liquid and forced
with a high pressure pump into the column tube. This column
which acts as a stationary phase is packed with tiny particles
of chromatographic material that effects the separation. The
physical and chemical interactions between the mobile phase and
stationary phase lead to the separation of components. As they
elute from the column, they reach the detector that measures their
amount and provides an output called as “liquid chromatogram”.
The most commonly used detectors are variable wavelength
detectors, fluorescence detectors, mass spectrometer detectors,
NMR detectors and refractive index detectors.
Fast Protein Liquid Chromatography (FPLC) is alike HPLC
in principle, but the only difference between them is the amount
of working pressure applied by the pumps to the column. FPLC
columns can only be used up to maximum pressure of 3-5 MPa
(435-580 psi) and in HPLC, the pressurized pump generates
pressure of 0-550 bar (14.6-8000psi). But the advantage of
the HPLC system is that it can use both the columns whereas
FPLC works only with the FPLC column .And systems that
operate at pressures < 50 psi (~3 bar) are characterized as Low
Pressure Chromatography (LPLC ) systems and are often used for
simple protein separations that do not require high resolution.
Larger particle size phases form the basis of low-pressure liquid
chromatography in which the flow of eluent through the column
is either gravity-fed or pumped by a low pressure pump, often
a peristaltic pump. This technique separates proteins through
a bed of porous beads where the molecules are eluted off the
column in order of decreasing size .
Proteins are fractionated from the cell lysate by using
different types of HPLC columns such as size exclusion, affinity,
ion exchange, ligand-exchange, reversed-phase, normal phase.
Separation mechanisms are chosen according to the size,
binding affinity, polarity, hydrophobicity of bio molecules. As
HPLC techniques are classified into various types according to
the nature of stationary phase, few of the most commonly used
chromatography approaches are reviewed here. Coupling MS
with multidimensional electrophoretic and HPLC techniques
enables the characterization of complex protein mixtures with
Reversed Phase-High Performance Liquid
Chromatography (RP- HPLC)
Segregation of molecules depending upon their hydrophobicity
for closely identical sequences of polypeptides of a wide range,
from small peptides obtained through trypsin digestion to large
proteins is possible through RP-HPLC. This technique is often
used because of its high productivity and resolution among close
related molecules along with good eproducibility of separations
done over longer periods of time. By changing the mobile phase
characteristics, chromatographic selectivity can be obtained.
The separation depends on the hydrophobic binding of the
solute molecule and the immobilized hydrophobic ligands where
proteins carry both hydrophilic and hydrophobic amino acids.
When the mixtures of proteins pass through the column, polar
proteins elute first followed by less polar and the remaining nonpolar
proteins bind to the column. Elution of bound hydrophobic
protein is facilitated by enhancing the concentration of organic
solvent, thereby increasing the retention time. Reverse phase
chromatography as cited in [55, 57, 58] is usually done alongside
mass spectrometry in a way to analyze the protein that is eluted
from the column.Wall et al. described a two-dimensional
liquid-phase separation method coupled with a MALDI-TOF mass
spectrometer to separate and profile the cellular proteins, by
using isoelectric focusing in the first dimension and RP-HPLC in
the second dimension and the selected fractions were analyzed
using MALDI-TOF MS. By using this technique, the authors
were able to resolve 700 protein bands with a high resolution
and substantiated this approach as an alternative to 2-D gel
Two-Dimensional Nano high –Performance liquid
For better separation and hydrophobic peptide recovery,
2D nano HPLC is used, specifically for complex peptides made
from enzymatic digests of selected proteomes. The peptides are
separated by their electric charge state and distribution through
elution of the digested peptides from the Strong Cation Exchange
(SCX) column with injected salt solution plugs of increasing
concentration. After which the eluted peptides are trapped and
introduced into RP nano flow path for separation to be done
based on hydrophobicity and are pre-concentrated before
introducing them to MS/MS analysis. The procedure is continued
until all peptide fragments are eluted and analyzed . Using
the combination of SDS-PAGE assay and two-dimensional nano
liquid chromatography (2D-nano LC) coupled online with tandem
mass spectrometry, the membrane proteins from mouse brain
tissues were detected and categorized in a research study led by
Van Chi and Nguyen Tien Dung . In another study, Choi et
al.  compared two methods, 2-DE coupled to MALDI-TOF-MS
and ESI-MS/MS analysis (2DE-MS) and the on-line 2D nano LC,
followed by nano ESI-MS/MS analysis (2DLC-MS) to identify and
characterize the proteins depleted from human plasma. Though
both the techniques identified a unique set of proteins, 2DLCMS
assay was more advantageous as it assessed the proteins in
a high-throughput manner.
Affinity chromatsography takes the advantage of unique
biological features of proteins to achieve separation and
purification based on specific protein-ligand interactions. But
this method requires that a detailed underlying characteristics
of the structure and biological specificity of the compound
in consideration to be known in order to achieve separation
conditions. As the protein solution is added, the ligands
immobilized on the column such as antibodies, receptors, ligands,
or specific binding partners bind specifically to the component to
be purified from the mixture. Mass spectrometry is then carried
out on the eluted protein from the column after it is washed
thoroughly to remove any non-specific bound proteins .
Reactive dyes in the column are also used in affinity
chromatography to separate and purify a wide variety of proteins
in a selective and reversible mode. Travis et al.  discovered
the ability of sepharose blue dextran conjugate to bind the
proteins of serum or plasma albumin in the column and the
results showed 96% of albumin adsorption to the SBD column.
Thomas and his coworkers adapted a similar approach for the
separation of albumin proteins from the blood plasma of four
species; rats, guinea pigs, baboons and humans and reported
highest percentage of recovery of serum albumin in human and
baboon than guinea pig or rat. Bécamel et al. discovered a
sensitive method to identify the interaction of multi protein
complexes with intracellular domains of membrane-bound
receptor (G-protein coupled receptors), by using peptide affinity
chromatography to purify the proteins. The eluted proteins
from the column were resolved on a high resolution 2-D gel and
stained with silver and further analyzed by MALDI-TOF mass
spectrometry. Also immunoblotting was combined along with MS
in order to detect low amounts of protein components
Ion Exchange Chromatography
The net charge on proteins at a particular pH varies due to
variations in the contents of charge damino acids .Ion exchange
chromatography is based on the reversible adsorptiondesorption
of ions in solution on a charged solid matrix or
polymer network. A positively charged and a negatively charged
matrix bind to anions and cations and are called anion-exchanger
and cation-exchanger respectively. Chemical modifications of the
matrix such as weak or strongly acidic and weak or strong basic is
possible since weakly acidic groups behave as cation exchanger,
and that with a weakly basic group behave as anion exchanger
which are appropriate for separation and purification of most
proteins. To ensure good bonding of protein to the ion exchange
column, the PH and ionic strength of elution buffer are optimized.
Contaminated proteins bind less strongly and hence pass quickly
through the column. Finally, by changing the buffer solution to a
different pH and ionic strength which will favor desorption from
the column in order to elute it.For many protein purifications,
such as albumin and IgG, ion exchange column chromatography
matrices are employed. Strong Cation Exchange chromatography
(SCX) followed by RP-HPLC is one of the most widely used
separation technique to identify simple and complex mixture
of peptides. Han et al. described a 2-D separation technique
to characterize the proteins present in microsomal fractions
of naïve and in vitro–differentiated human myeloid leukemia
(HL-60) cells, using SCX followed by RP-HPLC coupled with a
micro capillary liquid chromatography–electrospray ionization–
tandem mass spectrometry (μLC-ESI-MS/MS).
Sizes-Exclusion Chromatography (Sec)
Also known as gel filtration chromatography as the name
suggests, works by segregating proteins based on their molecular
size by determining their molecular weights and weight
distributions. The main advantage of this method is its ability to
maintain the native structure and function of the purified protein
since a large variety of buffers can be used to obtain the desirable
condition for the protein. This method works by adding a protein
solution into a stationary column packed with porousdextran or
agarosehydrophilicgel beads. When the protein passes through
the gel beads, the smaller molecules which can enter the pores
are slowed down whereas the bigger molecules which cannot
enter the beads, slide and move around the beads there by
rapidly passing through the column. Thus, molecules are eluted
off the column in order of decreasing size by varying the average
pore size of the beads which are available for a wide range of
molecular masses. The eluent is collected in fractions and later
analyzed by spectroscopic techniques .
In one of the studies, Opiteck et al.identified the
proteins from a bacterial cell lysate by employing the 2-D HPLC
system, where the proteins are separated using size-exclusion
chromatography (SEC) in the first dimension, and analyzed by
RP-HPLC based on their hydrophobicity in the second dimension.
The fractions selected from RP-HPLC column were then resolved
by MALDI-TOF/MS or ESI/MS. Also in another study, Opiteck et
al.  applied 2-D SEC/RPLC method coupled to electrospray-
MS to analyze a mixture of peptide fragments resulting from the
digestion of a protein. Owen et al. compared size exclusion
high-performance liquid chromatography (SEC-HPLC) with an
automated Immune Turbidimetric Assay (ITA) to evaluate the
amount of micro albuminuria (urinary protein). In these studies,
the results indicated that HPLC gave albumin concentration
nearly 4 times greater than those obtained by immunoassay,
which clearly suggests that SEC-HPLC is an appropriate method
to estimate the albumin proteins that are not detected by ITA.
Protein is an indicator of biological entity or activity. As
many of the medical conditions are associated with protein
malfunction, there is a need to isolate pure proteins and explore
various aspects of its structure and functions in order to design
diagnostic and the rapeutic tools for disease control. Hence
protein analysis is highly required in many research and clinical
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