Sample pretreatment (3)

After we extracted protein by various methods introduced in the last note, this matter is not over yet, because we need to carry out quality control on the extracted protein to confirm whether enough protein has been successfully extracted and whether there is pollution.

As shown above, quality control is divided into two parts:

Content determination: Check whether enough protein has been extracted. Pay attention to the incompatibility mentioned in the above picture. If SDS is added to the sample, BCA method can be selected to determine protein concentration instead of Bradford method. On the other hand, if reducing agent is added to the sample, protein can be determined by Bradford method instead of BCA method.

SDS-PAGE: Detect the extraction efficiency of protein and whether there is pollution. For example, we have 50 samples, but almost no bands can be seen, which indicates that the quantization is not accurate. If we want to find the differential protein from several groups of samples, it is particularly necessary to do SDS-PAGE to detect the extraction efficiency of protein in similar samples.

Taking the above figure as an example, the band in the middle of sample 0 is missing, which may be the difference caused by insufficient protein extraction or the difference of the sample itself. We can extract it again, and first rule out the difference caused by extraction; If it is the difference of samples themselves, it is suggested to use the unmarked method, and each sample should be quantified separately, instead of using iTRAQ or TMT to mark the quantification, otherwise the quantification will be inaccurate due to the influence of high-abundance proteins in the middle.

Let's look at several common problems and their solutions, as shown below:

Situation 1: The extraction results are quite different. This situation needs to be re-extracted, and whether it is the difference caused by insufficient extraction or the difference of the sample itself is detected;

Case 2: The molecular weight mark is on the left and the actual sample is on the right. It can be seen that there are few bands in the actual sample, which may be due to insufficient extraction. It is necessary to reset the extraction parameters and use stronger conditions and longer time to extract again;

Situation 3: There are many horizontal stripes and vertical stripes, which may be the influence of nucleic acid or lipoprotein. In this case, desalting treatment is needed, that is, the desalting column is combined with peptide, but not with other substances, so as to achieve the purpose of removing pollution.

Situation 4: The two bands are very similar, but one is obviously brighter than the other. What are the possible reasons for this? In the first case, because these samples belong to the same type, such as mouse muscle tissue, protein extraction of one sample is enough, while protein extraction of the other sample is not enough, so two different bands will be produced. In this case, it is necessary to re-extract. Another possibility is that, considering the SDS-PAGE with the same sample size, if the protein content displayed by the two bands is very different, it may be caused by the inaccurate determination result of the reference content, and it needs to be re-quantified.

Desalination

After protein extraction, desalination is needed. Let's see what methods can be used to achieve this.

Ultrafiltration: protein molecules of 10kDa and above can be intercepted, which is suitable for small samples. The operation steps can be: ultrafiltration concentration 100μL to 40μL, adding buffer to 100μL, ultrafiltration to 40μL, and repeating several times. In fact, it is difficult to completely remove pollutants (such as SDS) by ultrafiltration, and there will still be a very small amount of pollutants in the end, but when the concentration of these pollutants drops to a certain extent, the purity of the sample is considered acceptable.

Tips:

The concentration of urea in the sample should be controlled below 1M to avoid affecting the sample. When we extract protein, we use 8M urea. If we dilute it 8 times directly, the sample size will be too large. After adding enzyme in the next step, the concentration of enzyme and protein will be particularly low, which will greatly affect the enzymolysis effect. In addition, it is too big to handle. At this time, ultrafiltration can also be used for multi-step dilution to reduce the urea concentration to below1m.

Dialysis: protein molecules with 10kDa and above can also be intercepted, which is suitable for large samples, such as urine, in which salt can be dialyzed into external dialysate.

Propanol precipitation: acetone (V sample: V cold acetone = 1: 3 or more) is precipitated at -20℃ for more than 2 hours.

C 18 column desalination method: XBridge C 18 column produced by Waters Company uses the packing on the column to combine with protein, and the salt substances flow through it, so as to achieve the purpose of separation.

Reductive alkylation and enzymolysis

After desalination, we will take a very important step: reductive alkylation and enzymolysis. During the whole process, everyone watched the following picture:

There are two things I want to talk to you about first.

First of all, let's talk about why acetone precipitation should be placed after alkylation in the first step. Through the previous research, we know that acetone can dissolve the detergent and reducing agent of the sample, but protein will not dissolve in acetone, but will precipitate, thus achieving the purpose of removing impurities.

After alkylation, the globular protein becomes chain-shaped, and then the pollutants such as urea or SDS are removed by acetone precipitation, and then re-dissolved (that is, re-dissolved for the next enzymatic hydrolysis operation), then the chain-shaped protein will have a better re-dissolution effect than the globular protein, and the loss caused by insufficient re-dissolution can be avoided. Therefore, acetone precipitation should be carried out after alkylation.

In addition, regarding the restriction enzyme digestion step, if some antibodies are digested by trypsin only, because there are too few restriction enzyme digestion sites, the cut peptide segment is too long, which is not convenient for mass spectrometry detection. In this case, other enzymes, such as Lys-C, can be combined for multienzyme digestion to shorten the peptide segment. After testing, we found that Lys-C+ trypsin digestion can improve the identification rate 10%-20% compared with trypsin digestion alone.

Enzymatic hydrolysis needs to be completed in a buffer system, such as 25mM ammonium bicarbonate system (volatile, pH 7-8) or TEAB (triethyl diethylamine salt, 10- 100mM).

Tips:

If iTRAQ (or TMT) label is used, it is best to use TEAB instead of ammonium bicarbonate system. Because iTRAQ (or TMT) reagent is terminal amino group, the amino group on ammonium bicarbonate will also be labeled, which will affect the labeling efficiency of protein.

The dosage of enzyme can refer to the following formula:

W (enzyme): w (substrate) =1:20–1:50.

In addition, we should pay attention to the compatibility of pancreatin hydrolysis. Pancreatin can only tolerate urea 1M at most, and cannot be used with SDS.

Pre-fractionation of protein and peptides

As mentioned earlier, the mass spectrometer is an ion saturation instrument. The existence of high-abundance protein will suppress the signal of low-abundance protein, and the mass spectrometer will take some time to react. For example, human cells usually express 20,300 kinds of protein, and each kind of protein will produce 10-20 kinds of peptide segments after enzymolysis, so there are hundreds of thousands of peptide segments, and it is difficult to detect so many kinds of peptide segments at the same time by mass spectrometry. Therefore, the classification of peptide mixtures can reduce the difficulty of detection and get more peptide/protein identification results.

We can separate from protein level and peptide level, and combine various separation methods. Are you familiar with the level separation from protein? Usually, we use SDS-PAGE or IEF technology to separate and mix protein by taking advantage of the differences in physical and chemical properties of protein, such as molecular weight, shape and isoelectric point.

The second separation scheme is carried out at the level of peptide segment, and different fillers are used for separation according to the different properties of peptide segment.

SCX (strong cation exchange) is a strong cation exchange column based on silica gel, which can be combined with cations and eluted by ion exchange in buffer, so as to achieve the purpose of separation from other peptide segments without cations.

SAX (Strong Anion Exchange): A strong anion exchange column bonded with quaternary ammonium groups by silica gel can be combined with anions, and the anions are separated and eluted by ion exchange in buffer solution, so as to achieve the purpose of separation from other polypeptides without anions.

Rplc (reversed-phase liquid chromatography): a reversed-phase liquid chromatography column, which is different from a normal-phase column in that polar functional groups are bonded to its surface. For example, the surface of the reversed-phase column is bonded with nonpolar functional groups, which is called C 18 column. Other commonly used columns are C8, C4, C2, etc. Here, we choose C 18 column to achieve the purpose of separation according to the different hydrophobicity of peptide segments.

Hilic (Hydrophilic Interaction Liquid Chromatography): Hydrophilic chromatographic column can be used to separate polar compounds. It is difficult to separate strongly polar peptides because of their poor retention in reversed-phase chromatographic columns. Hydrophilic chromatographic column can be used to fix strongly polar peptides, and it can be combined with the mobile phase composed of high proportion of organic phase and low proportion of water phase to achieve the purpose of separation. This mobile phase composition is especially beneficial to improve the sensitivity of electrospray ionization mass spectrometry (ESI-MS).

High pH- low pH RPLC: The liquid phase condition of pH10 is combined with the acidic condition of RPLC pH2 for separation.

Tips:

Usually, we combine RPLC with mass spectrometry. Because RPLC system uses water and acetonitrile, which is volatile and salt-free, it can be directly sent to mass spectrometry for detection. However, a normal-phase chromatographic column like SCX/SAX needs to elute samples through a high-salt system, so it is not compatible with mass spectrometry. When we do multistage separation, all kinds of salts will elute in front of us, and then RPLC is the last one, and then mass spectrometry can be directly connected.

Multi-dimensional separation: for example, separation at protein level first, then separation at peptide level, or combination of multiple peptide levels. Next, we will focus on various strategies and effects of multidimensional separation.

Let's take a look at the picture above. Figure A in the upper left corner shows that the sample is divided into 40 fractions by high pH- low pH RPLC, and then combined into 20 fractions. This combination can make the peptide distribution in the sample more uniform.

The "Figure B" in the upper right corner shows the separation by SDS-PAGE, which is also divided into 20 fractions, and then combined into 5 fractions and 6 fractions by two combination schemes, also in order to make the peptide distribution of the sample more uniform.

Diagram C in the lower left corner compares two separation strategies of high/low pH RPLC and SDS-PAGE for the same sample. It is found that 5408 protein can be identified after the two separation methods, 195 1 protein can only be identified in the separation strategy of high/low pH RPLC, and SDS-PAGE can be used for separation. From this picture, the two methods are complementary.

The four small pictures in the lower right corner show that when we do classification and separation, the more grades we divide, the more protein we can identify. However, this growth is not linear. When the number of grades reaches a certain level, the number of identifiable protein will increase to saturation. Therefore, in the case of saving time and effort and ensuring the effect, you can choose the appropriate series.

Let's look at the number of protein that can be identified by multistage separation in published literature.

20 13 A document published on MCP reported that RP-RPLC was used for two-stage separation, which was divided into 24 conventional fractions, and the loading amount was100μ g g. More than 8,000 protein could be detected in one day.

A document published by Nat Comm 20 13 reported that it was classified by RP column first, then separated by SAX, and then separated by reversed-phase chromatography, with long column1m. The sample is human embryonic stem cells, and the loading amount is still100μ g g. After 8 days of online separation, 98 18 proteins were detected. If the separation time is extended to 24 days, 13075 protein can be detected.

A document published by Nat Method 20 14 reported that IEF (isoelectric focusing) combined with RPLC was used, and the sample was human epithelial cancer cell, with the loading amount of 800μg, which was divided into 360 fractions, and it took more than 15 days, and was analyzed by * * 13078.

As mentioned earlier, the more separation stages of protein and peptide, the more protein can be identified. However, due to the limitation of time and time, this trend will always be saturated to a certain extent, so we usually choose. For example, the conventional score of 10 can basically identify 5000-8000 protein. If it is a serum sample, it can be divided into more fractions, especially one-dimensional RPLC. If it is divided into 40 or 60 scores, it will be combined into 10 or 20 scores, which is about 30% more than the protein that can be identified by directly dividing into 10 or 20 scores!

Tips:

For fractionation, C 18 chromatographic column is usually used for fractionation, and there is no kit for this purpose.