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Nano Flow Liquid Chromatography
INTRODUCTION
The field of proteomics has experienced a fast evolution in recent years. Unlike the analysis of nucleic acids, there is not an equivalent method to polymerase chain reaction for the amplification of proteins. Thus the methodologies for proteomics have to be successfully sensitive to be able to detect endogenous levels of proteins. Due the analytical demands of biological samples, miniaturised liquid chromatography (LC) techniques were developed to allow for analysis of samples with greater sensitivity than that afforded by conventional LC, one of these methods, namely nano flow LC, with emphasis on studies that used it in combination with MS/MS.
PRINCIPLE
The main principle used in nano flow LC using flow rates in the range of low nano litre per minute which result in high analytical sensitivity due to the large concentrations efficiency afforded by this type of chromatography.
Based on the separation, the nano LC equipment was divided into the following:
- One-dimensional separation nano LC
- Two-dimensional separation nano LC
The main differences between these two separations are only in the column:
- In one-dimensional separation nano LC, the column was 75-m × 12 cm capillary column packed with 5-m porous C18 particles were used.
- In two-dimensional nano LC, the column was SCX-RPLC 2 D which is a biphasic column prepared by packing C18 and SCX particles sequentially into a fused silica nano tip was used.
INSTRUMENTATION
- Solvent delivery system: In solvent delivery system, the solvent flow was monitored by splitters and sensors.
- Splitters: Flow rates in the nl/min range have been achieved by using HPLC pumps operating at a relatively large flow rates and then splitting the mobile phase to obtain the desired flow. The primary flow rate was divided into the nano flow and waste flow.
- Active flow splitting with electronic sensor
- Sensors: To control the active splitting ratio, the nano flow was monitored with a nano flow sensors. This consists of a stainless steel capillary. The heat transport is proportional to the flow rate. The flow rate was controlled by the switching valves.
thermal flow sensor - Thermal flow sensor
- Columns: The mainly used columns in nano flow liquid chromatography are capillary columns. They are slurry packed to a length of 5–15 cm, although the use of long capillary packed to a length of 80-100 cm in combination with extended gradients afforded greater resolution and peak capacities than those achieved with shorter columns. Another trend to increase chromatographic resolution is to construct columns with stationary phases consisting of particle sizes of less than 2 μm.
As sample amounts and volumes continue to become smaller, the reduction of column id is the answer to obtain highest mass and detection sensitivity.
SAMPLE INJECTION
The sample was loaded by using switching configurations. In this, a short, larger bore trapping column was placed in the sample loop and analyte molecules are injected onto this column.
Another method was vented column principle. In this, a short concentrating column and the analytical column are connected by a T-junction.
Flow diagram of the nano flow chromatography instrument
DETECTORS
- Flow cells for UV-detectors have been developed with U and Z-shaped configurations that minimise dead volumes while maintaining a relatively long optical path.
- Mass spectrometers are most powerful detectors for nano LC and coupling nano LC to MS and nano LC-MS/MS. Low zeptomole (10−21 moles) detection limits have been reported for nano LC-MS when high-resolution chromatographic and mass spectrometric systems were combined when using nano ESI emitters.
COMPARISON BETWEEN WELL ESTABLISHED ON-LINE TWO-DIMENSIONAL NANO LC-MS AND OFF-LINE TWO-DIMENSIONAL NANO LC-MS
The comparison of the two-dimensional on-line LC-MS methods working with the injected salt solution plugs in increasing concentration (or) with the pumped semi-continuous gradient and off-line two-dimensional LC-MS method working with a continuous salt solution gradient in the first dimension a whole cell lysate from yeast (Saccharomyces cerevisiae) was analysed. In general, more proteins with higher score and sequence coverage could be identified with the off-line two-dimensional approach.
Table shows the comparison of the nano flow LC methods.
Required amount of protein/peptide in microcolumn separations is as follows:
Conventional LC
|
∼10,000 fmoles
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Micro LC
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∼1,000 fmoles
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Capillary LC
|
∼100 fmoles
|
Nano LC
|
∼1 fmoles
|
ADVANTAGES
- Nano flow LC-MS together with MALDI-IMS, the levels of PEP-19 and its special distribution Parkinsonism tissue was determined and compared to control.
- It was used for the better monitoring of therapeutic effect and tonicity.
- It was mainly used in the analysis of digested proteins and peptides.
- Example: 52 and 816 protein entries were identified from digested human IPI proteins.
- 892 and 584 peptides were identified from the trypsin digest in silica.
- Nano flow LC-Ms is used in identification of metabolite spermidine which gives detailed study amount of enzymes which are involved in regulated study of hyper thermophiles.
- Nano flow LC-Ms is used in the identification of BT-474 and MCf-7 cell from the combination of trypsin and staphylococcus V8, protease which give hydrophilic peptide from membrane proteins.
APPLICATIONS
- The main application of nano LC-MS was to remove high abundance proteins efficiently.
- Nano LC-MS was mainly used in the biochemical research, drug discovery, nutrition science and clinical practices.
- Used in analysis of peptides in serum.
- Example: Used for the analysis of neuropeptide Y in serum.
- It was used in analysis of small peptides in the urine of renal Fanconi patients.
- Nano LC-MS was used to sequence major histocompatibility complex peptides from cells infected with the measles virus and from uninfected cells.
- Nano LC-MS was also used to identify peptides from trypanosome cruzi processed and presented by the MHC class 1 pathway.
- It is used in the application of receptor affinity, gene knocking, antisense blocking and functional studies by administration of each peptide.
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