Protein identification using nanoscale liquid chromatography-tandem mass spectrometry
Tandem mass spectrometry is an effective technique for peptide characterization based on the fragmentation pattern of the peptide, also known as MS/MS scanning. In tandem mass spectrometry, each peptide fragment can obtain its own spectrum, which allows the identification of proteins based on peptide properties. This experiment is based on the "Guide to Plant Proteomics Experiments", edited by H. Thielemant, M. Zivi, C. Damerweiler, and V. Mitschine (France).
Operation method
Protein identification using nanoscale liquid chromatography-tandem mass spectrometry
Materials and Instruments
Modified Trypsin Move 3.1 Hydrolysis of endogelatin and extraction of peptides (one-day experimental protocol) For more product details, please visit Aladdin Scientific website.
Digestive Buffers Extractive Solutions Wash Solutions Decolorizing Solutions
Multichannel Pipette Guns Eppendorf Repeater Dispensers Combitips Dispenser Tubes
For proteins obtained from SDS-PAGE, 2D-PAGE, and BN-PAGE electrophoresis (stained with Kaumas Brilliant Blue G-250) [11], this protocol is feasible and effective. It does not require the use of an automated zymography (digestion robot). Using a multichannel pipette gun and microplate format, 96 samples can be processed in a day. The standard method does not require the reduction and alkylation of proteins. These two reaction steps are time-consuming but have no effect on most protein identifications, even for samples obtained by SDS-PAGE and BN-PAGE. However, based on our experiments, these two reaction steps seem to be an important source of keratin contamination. However, in the case of cysteine-rich proteins, protein reduction and alkylation may increase protein coverage (see Note 1). A multi-channel pipette gun is used to remove different liquids and an Eppendorf dispenser is used to dispense solutions. Protein reduction and alkylation should be carried out at 37°C with agitation.
( 1 ) Place gel cuts (1.5 mm diameter) into tubes. Non-liquid gels can be stored at 4°C for several weeks or at -20°C for several months. Seal with painters tape.
( 2 ) Decolorize: treat with 50 μl of Decolorizing Solution for 2 X 30 min.
( 3 ) Washing: Wash with 50 μl of washing solution for 15 min.
( 4 ) Shrinkage: treat with 50 μl shrinkage solution for 15 min.
( 5 ) Washing: Wash with 50 μl of detergent for 15 min.
( 6 ) Shrinkage: treat with 50 μl of shrinkage solution for 15 min.
( 7 ) Shrinkage: Treat with 50 μl of shrinkage solution for 15 min.
( 8 ) Place the strip holders on ice for 10 min, add 5 μl of trypsin (20 ng/μl of digestion buffer), and allow to stand for 10 min. then place the strip holders in a hot mixer for 4 h. After 2 h of trypsinization, add 5 μl of digestion buffer to compensate for evaporation of the liquid.
( 9 ) Extraction 1: Add 15 μl of extraction buffer and wait for 15 min. transfer the extract.
( 10 ) Extraction 2: Add 15 μl acetonitrile, wait for 15 min. transfer the extract.
( 11 ) Drying: evaporate overnight at room temperature or dry for 30 min using Spped VAC (see Note 2).
Optional Steps: Reduction and Alkylation
( 1 ) Reduction and alkylation can be carried out by treatment with 10 mmol/L (1.5 mg/ml) DTT and 55 mmol/L (10 mg/ml) iodoacetamide (IAA) (dissolved in 50 mmol/L NH4HCO3 ).
( 2 ) After completion of step 4 in 20.3.1, add 25 μl of 10 mmol/L DTT. react for 1 h at 56°C.
( 3 ) Remove the liquid and add 25 μl of 55 mmol/L IAA. allow the reaction to continue for 45 min at room temperature in the dark. remove the liquid and proceed to step 5 in 20.3.1.
3.2 HPLC and ESI setup
( 1 ) The main problem with nanoLC is misalignment or bad tube cutting. This causes time delays and dilutes the liquid. If there is dead volume between the 75 μm column and the mass spectrometer, a trailing effect can occur. If there is dead volume before the 75 μm column, peptides will elute as the retention time increases, but there will be no significant chromatographic degradation.
( 2 ) Before connecting, inspect the ends of the tubing with a magnifying glass to make sure they are clean and cut vertically.
( 3 ) Figure 20-1 gives a schematic of a nanoLC equipped with an RP trap column and a nanocolumn, as described in the following steps. Liquid connectors are used to provide voltage. This method extends the life of the jet needle (see Note 3).
( 4 ) Use 50 μm diameter tubing from the autosampler to the capture column, 50 μm diameter tubing from the capture column to the switching valve, 20 μm diameter tubing from the switching valve to the analytical column, and 20 μm diameter tubing from the analytical column to the spray needle.
( 5 ) Attach capillary tubing to valve ports with TEFZEL gaskets, TEFZEL rings, and PEEK nuts (standard port 1/16 in., 1 in. = 2. 54 cm). These fittings are lower pressure than stainless steel sealing washers and nuts, but are less likely to damage the capillary line when tightened.
( 6 ) The LC Packings nanoLC column is equipped with a 20 μm diameter outlet tube. The tube is cut into 3 cm (9 nl) pieces. The tube is connected to the nanoLC needle using a PTFE fitting (Teflon tube, 300 μm diameter), which is then shortened to 3 cm. This connection cannot tolerate the pressure when the needle is clogged. This disadvantage can also be an advantage: leakage of the PTFE coupling means that the tip of the needle is clogged, which is usually associated with irregular spraying.
( 7 ) Voltage is supplied using a liquid connector (Microtee connector). In our experience, this is the stronger configuration: the Microtee is placed in front of the 75 μm column and the electrode is a platinum wire (see Note 4). The spray needle with the liquid connector is uncoated (360 μm OD, 20 μm ID, 10 μm tip ID).
( 8 ) If the pump unit utilizes a shunt that only provides a flow rate of one nl/min without a flow rate detector (older versions of LC Packings instruments), the flow rate must be checked with a 25 μl syringe connected to a 75 μm column.The LC Packings system is a nanoLC and utilizes a 1: 1000 shunt. The pump flow rate should be 150 to 200 μl/min on a 75 μm column at 150 to 250 μl/min (see Note 5).
3.3 HPLC Separation
( 1 ) A typical 250 nl/min velocity gradient is given in Table 20-1. The process can also be optimized: e.g., the flow rate can be set to 300 nl/min during start-up and regeneration to minimize delays, and to 200 nl/min during peptide detection, which was developed and optimized in the peak packing system [12]. 
( 2 ) In part 1, from 0 to 3 min, the valve is in position 1-2 (see Table 20-1 and Figure 20-1). The autosampler injects 5 μl of sample, which is loaded and captured on the reversed-phase precolumn at a flow rate of 7 μl/min. At the beginning of Section 2, the valve switches to position 10-1, connecting the capture column to the column. The sample is then eluted using a 250 nl/min nanoHPLC gradient using a reversed-phase precolumn. Part 3 is a regeneration step to clean the column.
( 3 ) The gradient depends on the column and its accessories. It should be adjusted for a control such as trypsinized product of bovine serum albumin (BSA ) (100-300 fmol injection).
( 4 ) The enzymatic product is dissolved in 10~15 μl of sample solution containing TFA. TFA is used as an ion-pairing reagent in the loading solvent because it better captures peptides on the precolumn compared to formic acid. In addition, formic acid is used as an additive to solvents A and B in HPLC because TFA strongly inhibits ionization.
( 5 ) Under these conditions, a complete process takes 45 to 50 min. Figure 20-2 shows an example of a chromatogram from a complex peptide mixture. The peptides were eluted at 11-35 min. the MS-based peak pattern is similar to the UV pattern, which allows one to examine the effectiveness and quality of the separation. the TIC of the MS/MS scan shows a high signal-to-noise ratio over the range of peptide separations. 
3.4 Mass spectrometer setup
( 1 ) With nanoLC, the peptide sample can be eluted in only 10-40 s. Thus, the instrument elutes the peptide sample in less than 10 s. The peptide sample can be eluted in less than 10 s. Thus, the instrument can complete a cycle (a verification scan in MS mode and an associated MS/MS scan, an ingestion survey scan) in less than 10s. Some of the steps described here are specific to LCQIT with software Xcalibur 1.3, but the main principle is the same for all MS/MS instruments: MS/MS is data-dependent, i.e., if a data peak is detected in MS mode, the MS/MS will automatically acquire data.
( 2 ) Xcalibur - An efficient acquisition method is the " Nth " method. This method is a succession of 3 scans: (1) a full MS scan (m/z 350~1900); (2) a ZoomScan (scanning the two main ions with higher resolution); and (3) an MS/MS of these two ions (see Note 6).
( 3 ) The ionization voltage at the liquid junction is set at 1.2 to 1.6 kV. If it is necessary to increase the voltage by more than 160 kV, this is generally an indication of a problem with the spray needle or the buffer. In this case, first replace the nanoflow spray needle and test it with 1.3 kV. If the problem persists, change the buffer (see Note 7).
( 4 ) The LCQ instrument has a cleavage parameter of 0.22, an activation time of 50 ms, and a collision energy of 35% to 45%. the decrease in Qz from 0.25 to 0.22 means that smaller peptides can be detected in MS/MS scans (see Note 8).
( 5 ) Dynamic exclusion can be used to prevent sequential MS/MS analysis of the same ion (see Note 9).
( 6 ) Exclusion lists are used to avoid noisy analysis. Some polysiloxane ions typically occur at masses of 371 Th, 445 Th, and 462 Th [13] .
( 7 ) Using LCQIT, the "Nth" method, and an acquisition time of 50 min, a raw file size of approximately 12 Mo was obtained.
3.5 Data processing and parsing
( 1 ) Data analysis is a 3-step process.
a. In the first step, MS/MS spectra were extracted from the raw files and filtered.
b. In the second step, the MS/MS spectra are compared with the theoretical MS/MS spectra obtained from the selected sequence database. For an experimental MS/MS spectrum, the mass of the parent ion is used to extract "isobaric" peptides from the database. If a specific enzyme such as trypsin is used, only those peptides that are specific enough to produce isobaric peptides can be extracted from the database. The software then converts the peptide sequences in the MS/MS theoretical maps according to the cleavage rules and the annotations proposed by Roepstarff and Fohlman [14] .
c. In the last step, the calculation of correlation scores between the experimental maps and the different series of maps obtained from the sequence database was performed. Here the data were analyzed using the BIOWORKS software (formerly Sequest). In this software, the correlation score is called XC.
( 2 ) In contrast to Edman sequencing, the sequences identified are not based on the actual detection of contiguous amino acids. Peptide identification is not possible if there is no exact sequence in the database for querying. Moreover, peptide identification is performed based on correlation with known sequences. Similar to other algorithms, BIOWORKS employs a scoring system to give the most probable peptide in each MS/MS spectrum [ 15, 16]. This means that an incorrect peptide sequence can also be an alternative sequence, which can lead to incorrect protein identification. To minimize this error, at least two MS/MS spectra should be used to identify two peptides of the same protein.
( 3 ) - Proteins are generally identified using the protein database of the model species that has been sequenced. For other species, EST overlapping cluster databases are also appropriate (see Note 10). Protein databases and EST databases are available on some FTP sites (see Note 11).
( 4 ) Databases can be preprocessed using a database indexing adjustment method. This feature can reduce query times by a factor of 10. Unfortunately, index tuning fixes cleavage specificity and authorizes mass modification. If the Index Adjustment database is selected, methionine oxidation ( + 16 ) can be set as a variable modification (i.e. methionine can be considered oxidized) (see Note 12).
( 5 ) MS/MS spectra are extracted based on the intensity of the TIC and the time interval of the time range. If peptides are eluted in the 20 to 40 min range, these values are used as parameters for the time range. The TIC intensity must be set according to the minimum TIC for the correct MS/MS spectra (LCQ is usually 1X105~5X105 ).MS/MS extraction can produce 50~200 MS/MS spectra.
( 6 ) Large Scale Sample Data Acquisition allows sample processing in batch mode. A computer with BIOWORKS installed (Pentium 4 processor and 512 MB DDR SDRAM memory) requires 1~2 s per spectrum for comparison using the SWISS-PROT database, and 1~2 min for nanoLC analysis to obtain results.
( 7 ) First, the SWISSPROT database was used. This database can check for keratin contamination and tryptic peptides, and the library contains annotated sequences.
( 8 ) Second, use a plant-specific database. If an EST database is used, the software will translate it into a protein database. Set the frame of translation to six possibilities (three forward and three reverse translation codes).
( 9 ) The corresponding results for the different samples are stored in the sample name directory. Select Multiconsensus result to get the result.
( 10 ) Peptide identifications are filtered according to the XC thresholds 1.4-1.7, 2-2.5 and 2.5-3 (corresponding to peptides with 1, 2 and 3 charges, respectively). Some incorrect peptide identifications are also kept in the list. They are usually noisy spectra. This explains the need for manual checking when only two MS/MS spectra are used for identification.
( 11 ) Remove proteins that were identified with only one MS/MS spectrum. If the protein was identified with two MS/MS spectra, manually check that the peak with the highest intensity in the experimental spectrum was not used in the match.
( 12 ) If "no enzyme" is selected as a query parameter and trypsin is used for enzymatic digestion, check that the candidate peptide is indeed a tryptic peptide. This check seems to be a relevant criterion and should unambiguously verify the identification.
( 13 ) Table 20-2 is an example of a BIOWORKS summary output from a nanoLC- MS/MS analysis of protein spots from a two-dimensional gel after in-gel tryptic digestion. nanoLC-MS/MS runs produce hundreds of MS/MS spectra, and fewer than 100 have sufficient intensity to be extracted and used in a database query. The EST database used for querying was not indexed or enzyme specific, and methionine oxidation was considered variable. XC thresholds were used to filter peptide identifications (XC ranges up to 1.5, 2, and 3). Nineteen MS/MS profiles that passed the filter were identified as tryptic peptides (Table 20-2). The participation of lysine or arginine at the C-terminus of the identified peptides increased the confidence of the match when no particular enzyme was specified for the search. The charge value is the charge of the parent ion. Most tryptic peptides carry two charges. However, three singly-charged ions can identify peptides with significant XC values and confirm the identification results of doubly-charged gain ions (peptide DTGLFGVYAVAK) or identify new peptides (IDAVDASTVK and VTEEDVIR). Each MS/MS spectrum is associated with several peptides, but only the first hit is listed in Table 20-2. ΔCn is the difference between the correlation of the first and second hits (XC1- XC2)/XC1, which should be greater than 0.1. The value of the ions is the number of experimental ions in the MS/MS spectra that match the theoretical ions predicted from the peptide sequence. For example, 18/22 indicates that the MS/MS spectrum contains 18 of the 22 ions calculated. In the peptide sequence, M * corresponds to methionine sulfoxide; the oxidized methionine (+16 ) portion of the sequence is ILVANTAMDTDK and VVPEMVMAK. 