Multidimensional LC/MS/MS involves solution proteolysis of a complex mixture of proteins, which are then fractionated by high-performance liquid chromatogra-phy (HPLC). Peptides are then analyzed by tandem MS consisting of two phases. In the first phase, peptides in each chromatographic fraction are electrosprayed and ionized producing a mass spectrum characteristic of the molecular weight of each peptide in the sample. In the second phase, the first mass analyzer of the instrument is used to select a single (M + H)+ ion from the mixture and to transmit it to a collision chamber, where the peptide undergoes collisions with argon atoms and suffers fragmentation. The resulting fragment ions are then transferred to a second analyzer, which separates them according to mass . The end result is a mass spectrum containing ions characteristic of the sequence of amino acids in the selected peptide. When mixtures are extremely complex, online reverse-phase LC is used to concentrate and separate the peptides before sequencing by MS . An online capillary LC/MS/MS system consists of conventional HPLC pumps, transfer tubing, a precolumn flow splitter, a liquid junction, a reverse-phase microcapillary column, and a tandem mass spectrometer .
Two studies have used LC/MS/MS to identify differential protein expression in HIV- or HCV-infected cells. In the first study, traditional HPLC (ion exchange and reverse-phase columns) coupled to an ultrasensitive ion trap MS was employed to identify proteins that were unique to MDM and to identify proteins present in HIV-1-infected MDM lysates by microsequencing . The second study used normal hepatocytes and immortalized human hepatocytes that can be induced to express the entire HCV ORF. The two different cell types were labeled with an isotopically light (12C for stimulated) or heavy (13C, for control cells) reagent called isotope-coded affinity tag (ICAT); the two differentially labeled samples were then combined and digested with trypsin. Digested peptides were separated by strong cation-exchange chromatography, affinity purified with an avidin cartridge, and subjected to LC-ESI-MS/MS. This study led to the identification of 2159 unique proteins that could be used as markers for disease progression.
Some of the advantages include automation in sample application, ability to switch columns, and sensitivity, as this method is able to identify proteins at very low levels . In addition, this method has been extensively used for the determination of drugs and hormone levels in human serum [108-111], making it a promising tool for the detection of disease prognosis markers.
2DE-based proteome analysis provides information about protein abundance at the gel level by comparing staining intensities. However, when peptide mixtures are analyzed directly by LC/MS/MS techniques, the original quantitative information is lost. For this reason, one of the drawbacks of using LC/MS/MS is the dependence on incorporating stable isotope labeling for quantitative proteome analysis involving the addition of a chemically identical form of the analyte(s) containing stable heavy isotopes (e.g., 2H, 13C, 15N, etc.) to the sample.
SELDI-TOF is a proteomic technology that aims at the quantitative analysis of protein mixtures. This technique relies on the use of trapping surfaces that allow differential capture of proteins based on intrinsic properties of the proteins themselves to identify proteins from crude samples without the need for an initial separation step. A small amount of sample can be directly applied to a biochip coated with specific chemical matrices (hydrophobic, cationic, or anionic) or specific biochemical materials, including DNA fragments or purified proteins. Bound proteins can then be analyzed by MS to obtain either the protein fingerprints or the amino acid sequence when interfaced with a tandem MS.
The SELDI ProteinChip approach has been employed to study the protein profiles of cells infected with viruses, including severe acute respiratory syndrome coronavirus (SARS-CoV), HIV-1, and chronic hepatitis B virus infection (CHB) . SARS is a viral respiratory illness caused by SARS-CoV. SARS was recognized as a global threat in March 2003, after first appearing in Southern China in November 2002 (http://www.cdc.gov; ). Current serological methods used for laboratory diagnosis of SARS fail to guarantee early diagnosis since most are based on the detection of antibodies that are produced 17-20 days after the onset of symptoms. ELISA-based antigen detection tests offer high specificity and reproducibility, but they lack sensitivity. On the contrary, PCR-based methods, including reverse transcription-PCR, lack sensitivity and specificity . For this reason, there is a need to develop a diagnostic methodology that can detect SARS before the onset of the symptoms to allow for specific prevention and treatment measures for SARS. According to recent studies, SELDI-TOF seems to be a promising approach to study the protein profile unique for SARS. Sera from acute SARS patients or from healthy donors were examined to identify serum marker that could distinguish SARS from non-SARS patients. In this study, analysis of spectra accurately classified 36 of 37 (97.3%) SARS specimens and accurately classified 987 of 993 (99.4%) of the controls as non-SARS. In addition, the classification algorithm successfully distinguished acute SARS from other type of infections with very high precision . The same approach was also employed for the discovery of diagnostic pro-teomic signatures in the sera of patients with CHB having liver fibrosis and cirrhosis. Results show that 30 serum proteomic features formed a unique fingerprint for fibrosis that correlated with the different stages of fibrosis from minimal fibrosis to cirrhosis .
In another study that evaluated the protein fingerprints of HIV-1-infected MDM, cell lysates were directly applied on two types of protein chips: weak cation exchange and reverse-phase hydrophobic interaction. After washing to remove the unbound proteins, bound proteins were ionized and their molecular mass/charge ratio was determined using TOF analysis. Analysis of the obtained profiles showed distinct patterns between uninfected and infected MDM .
The SELDI ProteinChip approach allows for high-throughput protein analysis of crude protein mixtures without the need for a separation step. It is sensitive since it takes advantage of the analytical capacity of MS combined with novel surface chemistry. It can provide a phenotypic fingerprint of complex mixtures. Sample requirements are dramatically reduced, and because this approach employs MS
for its readout, attomolar to femtomolar concentrations of proteins can be detected. Additionally, reproducibility is greater than that of other techniques such as 2D gels; proteins at extreme pIs can be identified, a condition that is problematic under normal 2D gel electrophoresis conditions; and finally there is a greater sensitivity and accuracy for low-molecular-weight proteins (<25 kDa) using SELDI, especially below 10 kDa, which is particularly troublesome for 2D gels.
This method needs a very robust algorithm to ensure specificity of the profile, in that it can distinguish the pattern between disease and healthy individuals with high accuracy, taking into account variations in profiles between healthy individuals as well as persons with a variety of different infections at different time periods in their course of illness. Two additional drawbacks of this approach are the following: (i) The identity of the proteins cannot be discovered and (ii) as the absolute intensity of the peaks is measured in relationship to the most abundant peaks, peaks in low abundance will be masked by the more abundant ones. In addition, this method employs the direct analysis of tissues or biological fluids by MALDI. The main drawbacks of this approach are the preferential detection of proteins with a lower molecular mass and the difficulty in determining the identity of proteins owing to PTM obscuring the correspondence of measured and predicted masses.
3.4. Protein microarray: vaccinia virus
A protein microarray relies on high-throughput amplification of each predicted ORF by using gene-specific primers, followed by in vivo homologous recombination into a T7 expression vector. The proteins are expressed in an Escherichia coli-based cell-free in vitro transcription/translation system. The protein products from the unpurified reactions are printed directly onto nitrocellulose microarrays without further purification .
This approach was used to determine the complete antigen-specific humoral immune-response profile from infected humans and animals. The vaccinia virus proteome containing 185 individual viral proteins was printed on a chip after cloning and expression. The chips were then used to determine the antibody profile in serum from vaccinia-virus-immunized humans, primates, and mice .
Once it has been developed and produced, a protein microarray can be a very rapid method (3 days for most of the genes) to comprehensively scan the humoral immune response of vaccinated or infected individuals.
The generation of a complete proteome is technically challenging. One problem is the amplification of long genes. Furthermore, expression of some proteins in heterologous systems is not efficient. This technique also does not take into account PTM of viral proteins that are expressed in bacteria. Lastly, expression in E. coli might lead to folding problems of the protein.
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