Novel method for protein O-GalNAc modification rapid library search and deep coverage

Abstract

The invention discloses a novel method for protein O-GalNAc modification rapid library search and deep coverage. According to the novel method of the invention, on the basis of the extraction and classification strategies of the spectrograms of oxonium ions and sialic acid fragments in an O-GalNAc glycopeptide spectrogram, corresponding matched O-glycosyl glycoform database retrieval is carried out on different types of spectrograms, so that a database retrieval space is greatly reduced, and database search time is shortened; in order to solve the problem that a large number of missing valuesexist in the multi-sample detection of complete O-GalNAc glycopeptides under a mass spectrum DDA scanning mode, matching based on corrected retention time and a mass spectrum primary mass number is utilized to supplement the quantitative missing values of the complete O-GalNAc glycopeptides between different samples according to the complete O-GalNAc glycopeptides which have been identified in a multi-sample experiment, and therefore, the identification coverage of the O-GalNAc glycopeptides during multi-sample detection is substantially improved, and quantitative reproducibility is improved.

Description

technical field

[0001] The invention relates to the field of bioinformatics, in particular to a new method for rapid library search and deep coverage of protein O-GalNAc modification. Background technique

[0002] O-acetylglucosamine (O-GlcNAc) modification is a monosaccharide modification that occurs on acetylglucosamine linked to the hydroxyl ends of protein serine and threonine. O-GalNAc modification is an important protein post-translational modification, which plays an important role in the occurrence and development of various biological processes and diseases. Therefore, the analysis of protein O-GalNAc modification is of great significance for in-depth understanding of the nature of life activities and the diagnosis and prognosis of diseases. At present, protein post-translational modification research based on biological mass spectrometry is the most commonly used analytical method for O-GalNAc modification. However, there are major challenges in the analysis of ma...

Examples

Embodiment 1

[0053] Embodiment 1, urinary protein O-GalNAc spectrum classification search

[0054] Step 1. Urinary protein extraction and enzyme digestion

[0055]Take 10mL of the middle morning urine of healthy people, centrifuge at 12000g for 15 minutes to remove impurities, take the supernatant, transfer it to a 50mL centrifuge tube, add 3 times the volume of pre-cooled acetone, mix well, and then stand at -20°C for 2-4 hours for urine analysis. protein precipitation. After the precipitation is completed, centrifuge at 12000g for 15 minutes, collect the precipitated part, add lysate (8M urea, 0.1M Tris-HCl, pH=8.5) after drying, and use an ultrasonic breaker to sonicate urine protein (30% power, each time Ultrasound for 2 seconds, repeated 10 times), after the end of the ultrasound, centrifuge at 16000g for 15 minutes, and take the supernatant, namely the urine protein extract. Dithiothreitol was first added to the obtained urine protein extract to make the concentration 10mM (the fun...

Embodiment 2

[0064] Example 2. O-GalNAc Glycopeptide Matching and Quantitative Missing Value Filling Using Chromatographic Retention Time Correction and Accurate Charge-to-Mass Ratio

[0065] Step 1-step 4 is the same as embodiment 1.

[0066] Step 5. Chromatographic retention time correction and quantitative missing value filling

[0067] After searching the Byonic database and extracting information from the raw files of 16 healthy male samples and 20 healthy female samples, the retention time prediction of the identification results in 36 samples was carried out. The correction method is as follows: First, the O-GalNAc glycopeptides identified at least 3 times in 36 files (identified in at least 3 samples out of 36 samples) were identified as high-confidence peptides, and quantitative analysis was performed on them. A total of 487 high-confidence peptides were found, and the reference retention times of these 487 O-GalNAc glycopeptides were calculated (see above for specific methods). ...

Embodiment 3

[0069] Example 3. Using chromatographic retention time correction and accurate charge-to-mass ratio for O-GalNAc glycopeptide matching and quantitative missing value filling accuracy verification

[0070] Step 1-step 4 is the same as embodiment 1.

[0071] Step 5. Chromatographic retention time correction and quantitative missing value filling

[0072]The samples from the same case of urinary protein glycopeptides were divided into 5 equal parts, and then the mass spectrometry data were collected according to the method of step 4 of Example 1. Retention time prediction of identifications in samples. The correction method is as follows: First, the O-GalNAc glycopeptides with MS / MS information identified in the 5 files are identified as high-confidence glycopeptides, and quantitative analysis is performed on them. A total of 267 high-confidence glycopeptides were found, and the reference retention times of these 267 O-GalNAc glycopeptides were calculated respectively (see abov...

technical field

[0001] The invention relates to the field of bioinformatics, in particular to a new method for rapid library search and deep coverage of protein O-GalNAc modification. Background technique

[0002] O-acetylglucosamine (O-GlcNAc) modification is a monosaccharide modification that occurs on acetylglucosamine linked to the hydroxyl ends of protein serine and threonine. O-GalNAc modification is an important protein post-translational modification, which plays an important role in the occurrence and development of various biological processes and diseases. Therefore, the analysis of protein O-GalNAc modification is of great significance for in-depth understanding of the nature of life activities and the diagnosis and prognosis of diseases. At present, protein post-translational modification research based on biological mass spectrometry is the most commonly used analytical method for O-GalNAc modification. However, there are major challenges in the analysis of ma...

Examples

Embodiment 1

[0053] Embodiment 1, urinary protein O-GalNAc spectrum classification search

[0054] Step 1. Urinary protein extraction and enzyme digestion

[0055]Take 10mL of the middle morning urine of healthy people, centrifuge at 12000g for 15 minutes to remove impurities, take the supernatant, transfer it to a 50mL centrifuge tube, add 3 times the volume of pre-cooled acetone, mix well, and then stand at -20°C for 2-4 hours for urine analysis. protein precipitation. After the precipitation is completed, centrifuge at 12000g for 15 minutes, collect the precipitated part, add lysate (8M urea, 0.1M Tris-HCl, pH=8.5) after drying, and use an ultrasonic breaker to sonicate urine protein (30% power, each time Ultrasound for 2 seconds, repeated 10 times), after the end of the ultrasound, centrifuge at 16000g for 15 minutes, and take the supernatant, namely the urine protein extract. Dithiothreitol was first added to the obtained urine protein extract to make the concentration 10mM (the fun...

Embodiment 2

[0064] Example 2. O-GalNAc Glycopeptide Matching and Quantitative Missing Value Filling Using Chromatographic Retention Time Correction and Accurate Charge-to-Mass Ratio

[0065] Step 1-step 4 is the same as embodiment 1.

[0066] Step 5. Chromatographic retention time correction and quantitative missing value filling

[0067] After searching the Byonic database and extracting information from the raw files of 16 healthy male samples and 20 healthy female samples, the retention time prediction of the identification results in 36 samples was carried out. The correction method is as follows: First, the O-GalNAc glycopeptides identified at least 3 times in 36 files (identified in at least 3 samples out of 36 samples) were identified as high-confidence peptides, and quantitative analysis was performed on them. A total of 487 high-confidence peptides were found, and the reference retention times of these 487 O-GalNAc glycopeptides were calculated (see above for specific methods). ...

Embodiment 3

[0069] Example 3. Using chromatographic retention time correction and accurate charge-to-mass ratio for O-GalNAc glycopeptide matching and quantitative missing value filling accuracy verification

[0070] Step 1-step 4 is the same as embodiment 1.

[0071] Step 5. Chromatographic retention time correction and quantitative missing value filling

[0072]The samples from the same case of urinary protein glycopeptides were divided into 5 equal parts, and then the mass spectrometry data were collected according to the method of step 4 of Example 1. Retention time prediction of identifications in samples. The correction method is as follows: First, the O-GalNAc glycopeptides with MS / MS information identified in the 5 files are identified as high-confidence glycopeptides, and quantitative analysis is performed on them. A total of 267 high-confidence glycopeptides were found, and the reference retention times of these 267 O-GalNAc glycopeptides were calculated respectively (see abov...

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