Jia Wei
Waters Technology (Shanghai) Co., Ltd. Experimental Center
For trace and complex samples, such as proteomic samples, residual host cell proteins (HCPs) in protein drugs, etc., not only do they require a highly sensitive nanoscale liquid phase, but they also require more adequate separation. Online two-dimensional nano-liter separation technology (on-line 2D NanoLC) came into being, and has become an essential separation method for liquid analysis of trace complex samples.
The traditional nano-on-line two-dimensional technology generally uses strong cation exchange (SCX) as the first dimension and reversed phase chromatography (RP) as the second dimension separation method. This method is based on the orthogonal relationship between the ionic properties and hydrophobicity of the sample in the salt solution. However, SCX-RP technology is difficult in the separation of nano upgrades. The difficulty comes mainly from the SCX separation dimension. In the SCX separation, a higher concentration of salt solution is required as the mobile phase, but the salt-containing mobile phase is prone to salting out or causes the sample to precipitate in the pipeline, while the inner diameter of the nanoliter liquid phase is very small (25-100). Micron). Therefore, when the SCX-RP separation is actually used, the pipe blockage often occurs and the experiment fails.
To this end, in addition to providing traditional SCX-RP separation technology, Waters has creatively developed a two-phase two-phase separation method. (RP-RP). This RP-RP technology does not require the use of a high concentration of salt solution as the mobile phase, avoiding the pipe blockage problem easily caused by ion exchange separation, thereby greatly improving the system stability and practicability of the nanoliter two-dimensional liquid phase. What is even more exciting is that the comprehensive experimental comparison of Jarrod A. Marto of Harvard Medical School found that the liquid chromatography analysis obtained by RP-RP separation technique is better than the SCX-RP method (Fig. 1) [1] RP The -RP double-reverse two-dimensional method can help scientists get more protein analysis results.
This is because: 1. The salt buffer used in the SCX method is prone to ion noise background, which affects the quality of mass spectrometry data. 2. The separation effect of SCX depends on the number of charges carried by the polypeptide, while the amount of charge carried by the polypeptide is limited, so the first The dimensionality of the SCX is poor, resulting in low quality of liquid data information.
The R PR P dual-phase separation technique uses reversed-phase chromatography in both the first and second dimensions, so how does it achieve the orthogonality of the separation properties necessary for two-dimensional separation? It turns out that the reverse retention behavior of peptides is different under different pH conditions (Fig. 2) [2]. Based on this nature, Waters scientists have developed a unique RP-RP nanoliter online two-dimensional system - nanoACQUITY UPLC ® System with 2D-LC. The separation column of this system uses UPLC's consistent sub-two micron particle packing, so it has the advantages of UPLC's ultra-high resolution. In addition, it does not require a split flow to achieve a precise nanoliter flow rate, which saves the lab a huge purchase of high-purity mobile phase and waste disposal costs, and is more environmentally friendly. The advantages of the nanoACQUITY UPLC System with 2D-LC dual-phase two-dimensional system are summarized as follows:
â– More protein identification results can be obtained using the RP-RP system than SCX-RP technology.
â– The RP-RP system is more stable and durable than the SCX-RP system.
â– NanoACQUITY UPLC has superior separation of UPLC compared to nano HPLC.
â– Accurate nanometer without splitting
The nanoACQUITY UPLC System with 2D-LC dual-phase online two-dimensional system structure and analysis process is shown in Figure 3, including three columns: high pH reversed phase column, capture column, low pH reversed phase column. In this system, the first dimension column is a high pH column. After the sample enters the first-dimensional column, the first-dimensional gradient pump can automatically stepwise increase the organic phase according to the user's requirements to elute the different hydrophobic peptides in the sample in batches. The polypeptide eluted from the high pH reverse phase column is captured by the enrichment column. Each batch of enriched peptide will enter the low pH reverse phase analysis column in a linear gradient mode of the second dimension pump, where after sufficient separation, the sample will reach the ion source and enter the mass spectrometer.
The lower left figure is a schematic diagram of the structure. Step 1: After the sample is collected by the autosampler, it is pushed into the high pH column by the first dimension gradient pump. Step 2: The sample is washed out by a step gradient of the first dimension pump, and then enriched by the capture column. Wherein the second dimension gradient pump dilutes the solvent to a system suitable for capture column enrichment by applying 9 times the aqueous phase mobile phase of the first dimension pump. Step 3: After the six-way valve is switched, the second-dimensional pump is thoroughly separated by a linear gradient and sent to mass spectrometry. After step 1 is performed, steps 2 and 3 are alternated until the desired analysis is completed. The dual-phase online two-dimensional system nanoACQUIT Y UP LC System with 2D-LC has been widely used in the liquid chromatography analysis of peptides, helping researchers to obtain a lot of valuable research results.
Figure 3. Structure and analysis flow diagram for the nanoACQUITY UPLC System with 2D-LC system.
references
(1) Zhou F, Cardoza JD, Ficarro SB, Adelmant GO, Lazaro JB, Marto JA. Online Nanoflow RP-RP-MS Reveals Dynamics of Multicomponent Ku Complex in Response to DNA Damage. J Proteome
Res. 2010, 9, 6242-6255.
(2) Gilar M, Olivova P, Daly AE, Gebler JC. Two-dimensionalseparation of peptides using RP-RP-HPLC system with different pH in first and second separation dimensions. J. Sep. Sci. 2005, 28, 1694–1703 .
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