Bu Lina Yang Shuanying Du Jie Zheng Huadong Huo Shufen Shang Wenli Liu Yanfeng Lin beautiful rockAbstract: Objective To investigate the appl
Bu Lina Yang Shuanying Du Jie Zheng Huadong Huo Shufen Shang Wenli Liu Yanfeng Lin beautiful rock
Abstract: Objective To investigate the application of liquid chip mass spectrometry (CLINPROT) in the screening of serum marker proteins in patients with lung squamous cell carcinoma. Methods CLINPROT system comparison and analysis of 34 cases of lung squamous cell carcinoma (squamous cell carcinoma group), 46 cases of benign lung disease patients (positive control group) and 44 normal persons (normal control group) the expression of serum protein, FlexAnalysis3.0 software was used for data analysis, screening of differentially expressed proteins; by liquid chromatography tandem mass spectrometry (LC-MS/MS) biomarkers were identified. The results were compared with the normal control group of lung squamous cell carcinoma and squamous cell carcinoma of the lung benign group and control group serum protein expression profile in Z (M/Z) 800 ~ 10000 range, the former screened 96 differentially expressed protein peaks, M/Z protein peaks at 4054.13 and 4267.46 in the two group, the difference between the maximum and the ability to establish with the 2 kinds of different protein coordinates has good distinguish lung squamous cell carcinoma and normal people; the latter screened 99 differentially expressed protein peaks, M/Z protein peaks at 5065.27 and 4054.02 of the maximum difference between the two groups, the ability to establish with the 2 kinds of different protein coordinates has good distinguish lung squamous cell carcinoma and benign lung disease. M/Z 1778 and protein were identified by LC-MS/MS, and the results showed that both of them could be C3 fragments or C3 precursors. Conclusion lung squamous carcinoma patients and normal people, and between patients with benign pulmonary disease serum protein expression profile differences, application of liquid chip mass spectrometry technology from serum were screened with high sensitivity and specificity protein markers of lung squamous cell carcinoma.
Lung neoplasms; carcinoma; squamous cell; microarray analysis; mass spectrometry; tumor markers; diagnosis
Application of liquid chip-mass spectrometry technology for screening serum biomarker proteins in lung squamous cell carcinoma BU Li-na*, YANG Shuan-ying, DU Jie, NAN Yan-dong, LIN Xiu-li, ZHENG Hua-dong, HUO Shu-fen, SHANG Wen-li, LIU Yan-feng. *Department of Respiratory Medicine, Second Affiliated Hospital of Medical School, an Jiaotong University, Xi 'Xi' an 710004 China.
Corresponding author: YANG Shuan-ying Department of, Respiratory Medicine, Second Affiliated Hospital of Medical School Xi an Jiaotong University Xi "," an China, 710004, Email: firstname.lastname@example.org
[Abstract] Objective To screen the serum biomarker proteins of lung squamous cell carcinoma (SCCs) by liquid chip-mass spectrometry technology. Methods All serum samples including, 34 SCCs, 46 benign lung diseases (BLDs) and 44 healthy individuals, were compared and analyzed by CLINPROT system in order to study the serum protein expression profiles.
Project supported by the National Natural Science Fund (30570795); the Ministry of education program for New Century Excellent Talents Project (NECT-06-0845); Shaanxi province science and technology research project of [2007K09-01 (3)]; Xi'an city science and technology project of [SF08009 (1)]
Department of Department of respiration of Second Affiliated Hospital of Xi'an Jiao Tong University Medical College, 710004 (Bu Lina, Yang Shuanying, Du Jie, Lin Xiuli, Zheng Huadong, Dong Nanyan, Huo Shufen, Shang Wenli, Liu Yanfeng); the second hospital of Xi'an city (Bu Lina)
Correspondence Author: Yang Shuanying, Email: email@example.com
Then the discriminatory proteins were detected by FlexAnalysis3.0 software. Biomarkers were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) Results the differential serum. Comparing expression proteins between SCCs and healthy individuals, SCCs and BLDs, respectively, protein peaks [mass-to-charge 96 differential ration (M/Z) between 800 and 10 000] were found between SCCs and healthy individuals. In these protein peaks, the expression of protein peaks at 4054.13 M/ Z and 4267.46 M/Z had the largest difference between them. The two protein peaks could accurately separate SCCs from healthy individuals by the frame of axes Similarly differential protein peaks were, 99 automatically detected between SCCs and BLDs, In t. Hese protein peaks, the expression of protein peaks at 5065.27 M/Z and 4054.02 M/Z had the largest difference between them. The two protein peaks could accurately separate SCCs from BLDs by the frame of axes. Identified by LC-MS/MS, 1778 M/Z and 1865 M/Z might be assayed jointly and corresponded to complements C3 fragment or C3f precursor. Conclusions Differential protein expressions could be existed between SCCs versus healthy individuals and SCCs versus BLD patients. It is feasible to screen the diagnostic serum biomarkers of SCC with a high sensitivity and specificity by using CLINPROT system.
[Key Words] Lung neoplasms; Carcinoma; squamous cell; Microchip analytical procedures; Mass spectrometry; Biomarker; Diagnosis
Squamous cell carcinoma (SCC) is one of the most common histological types in lung cancer, and it is lack of effective biomarkers, especially early diagnostic value. Liquid chip mass spectrometry (MS) is a new method for proteomics research, and is a powerful tool for screening differentially expressed proteins and seeking new markers. The CLINPROT system technology developed by magnetic separation system, matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) based on the analysis of software and optional liquid automatic processing system composed of mass spectrometry in detecting system, and has high repeatability, can detect 2000 different polypeptides or proteins in the blood, and can the potential tumor markers were identified. In recent years, some significant results have been obtained in the early diagnosis of colorectal cancer, ,  and other diseases by using this technique. In this study, the application of this technique in the screening of serum marker proteins in patients with lung squamous cell carcinoma was discussed.
Objects and methods
1 lung squamous cell carcinoma group. 34 cases were selected from the Second Affiliated Hospital of Xi'an Jiao Tong University School of medicine and the second hospital of Xi'an from October 2003 to May 2008 of outpatient and inpatient patients. All the patients were diagnosed by histopathological examination, and were not treated with radiotherapy, chemotherapy and surgery before the blood collection. There were 22 males and females, aged from 44 to 78 years old, with a median age of 60 years (range) of 12 years. The TNM stage of the International Anti Cancer Alliance: 6 cases of stage B, stage II A stage in 2 cases, stage II B stage in 2 cases, stage A stage in 10 cases, stage III B stage in 9 cases, and stage IV in two cases.
2 benign pulmonary disease group (control group): a total of 46 patients with pulmonary benign diseases were selected from the two hospitals. Male 28 cases, female 18 cases; age ranged from 31 to 76 years old, the median age was 59 years; 12 cases of pneumonia, 11 cases of chronic obstructive pulmonary disease, 6 cases of pulmonary tuberculosis, 6 cases of pulmonary fibrosis, 4 cases of bronchial asthma, 3 cases of bronchiectasis, 2 cases of sarcoidosis, pulmonary cyst and inflammatory pseudotumor of the in 1 cases.
3 normal control group: a total of 44 patients were selected from the two hospitals in the same period. There were 26 males and females, aged from 44 to 76 years old, with a median age of 58 years (range) of 18 years.
All subjects were informed consent before the blood collection.
Two, the main material
Alpha cyano -4- hydroxy cinnamic acid (HCCA), WCX immunomagnetic (weak cation bead) and CLINPROT system purchased from Germany Bruker company Daltonics; 3K30 type low temperature centrifuge was purchased from Sigma company in Germany; NanoAquity UPLC liquid chromatography was purchased from American Waters company; LTQ Orbitrap XL electrospray mass spectrometer was purchased from ThermoFisher company in the United States; negative pressure storage vessel, pipettes, Eppendorf 3810 type polypropylene microcentrifuge tube (EP tube) and a gun head purchased from Xi'an Runde Biotechnology Co. ltd..
1 serum samples were collected: all subjects were collected fasting peripheral venous blood of 5 ml, 4 C standing for 2 h; 5000 r/min (centrifugal radius 5.5cm) centrifugation at room temperature 5 ~ 10 min; the supernatant was implanted 1.5 ml EP tube, 4 C and 10000 r/min (centrifugal radius 5.5cm) centrifugal 10 ~ 15 min; take the supernatant from 50 to 100 L into 0.5 ml EP tube, -80 stored at standby.
2 serum beads: 4 C refrigerated storage chamber to remove WCX immunomagnetic beads suspensions, fully mixing 1 min. 10 µ, 10 µ L cell; l phosphate buffer (PBS) with 200 µ l sample tube, then add 5 µ l of tested serum, mixing, room temperature for 5 min. The sample tube in the magnetic separator, magnetic attachment 1 min; absorb suspension liquid, avoid sucking magnetic beads; add 100 µ l beads; wash buffer; completely absorb suspended liquid. Remove the sample tube from the magnetic separator, and 5µ was added to the sample tube; l beads elution buffer, dissolved adherent beads. The sample tube in the magnetic separator, magnetic attachment 2 min, bead and suspended liquid fully after the separation of the supernatant into a clean 0.5 ml sample tube. 5µ l beads stable buffer, careful mixing for suction call, mass spectrometry or frozen.
3 points: freshly prepared HCCA, 0.3 g/L and 2/1 chromatographic grade ethanol / acetone. The sample tube was placed in the ice box for precooling. Bead elution buffer in preparation for mass spectrometry (anchor-chip target) with the target samples, and in a few seconds and magnetic separator, 10 matrix µ l mixed after suspension point target detection machine, drying at room temperature.
4 data acquisition and processing: the relative molecular mass error of CLINPROT system is corrected to < 0.1%. In the experiment, every 8 samples were collected before calibration. As a standard for calibration, 11 peptides were included, and the average relative molecular mass deviation was less than < 0.01%, the standard of using angiotensin. In the experiment, 13 standard serum was used as the quality control, and the variation coefficient was 30%. The parameters of instrument analysis: positive ion spectra, ion source acceleration voltage of 20 kV, delayed ion extraction 120 ns, 25 Hz laser frequency detection, the highest protein molecular weight was 20000. The optimization of data collection ranges from 800 to 10000, each sample collected from 400 point signal averaging. The original data is collected and processed by FlexAnalysis3.0 software. MALDI-TOF mass spectrometry data collected by Top Hat Baseline baseline processing and application of Savitsky Golay smoothing filter, filter the signal-to-noise ratio of < 4 peaks; corrected and normalized data, using peak area as a quantitative indicator of protein peak to average serum protein profiles.
To obtain the protein peak detection data were analyzed by Anderson-Darling test, 22 t test and Wilcoxon test, Anderson-Darling test, according to the P value of 22 t test P value and mean maximum and minimum values of the difference of the order for the detection of the value of the size and the distribution types of data are gradually comparison, calculate the difference protein peaks between groups, obtaining differential protein. According to the Wilcoxon test P value (PWKW) sorted protein peaks in front of the PWKW is smaller, the more significant difference between the 2 groups, the stronger ability of a single packet. Genetic algorithm (genetic algorithm) was used to evaluate the sensitivity and specificity of the differentially expressed proteins.
5 mass spectrometry identification: the serum samples of lung squamous cell carcinoma (SCC) were randomly selected and identified by liquid chromatography tandem mass spectrometry (LC-MS/MS) in the. Freeze dried samples were dissolved using a mobile phase A and the final volume was 20 L. Chromatographic column: capture column application Symmetry® C18, 5 m, 180 m x 20 mm, nanoAcquity™ Column; analytical column using BEH300 C18, 1.7 m, 75 m * 200 mm, nanoAcquity™ UPLC™ Column. The mobile phase A:5% aqueous solution of acetonitrile and 0.1% formic acid; the mobile phase B:95% aqueous solution of acetonitrile and 0.1% formic acid; trapping velocity 15 l/min, trapping time of 1.3 min, 300 nl/min flow rate analysis; analysis of 70 min; column temperature of 35 DEG C; Partial Loop model sample, sample volume 5 L. Nano ion source spray voltage of 2 kV; mass spectrometry scanning time of 60 min; experimental model for data dependence and dynamic exclusion ranges from 300 to 2000; scanning mass (M/Z); a scan using Obitrap, the resolution is set to 100000; CID (Collision induced dissection, collision induced dissociation) and two scan using LTQ Orbitrap 6; ion intensity is the strongest in the first selection of single isotope mass spectrometry as the parent ion MS/MS analysis (single charge exclusion, not as the parent ion). The use of data analysis software BioworksBrowser 3.3.1 SP1 Sequest™ IPI Human database retrieval, retrieval (3.45) , the parent ion error is set to 0.005%, the error of fragment ions of 1 as a way for non enzyme digestion. Search results Delta Cn = 0.10; Xcorr two charge 2.5, three charge 3, three charge for more than 3.5.
Comparison of 1 groups of average serum protein profiles of the FlexAnalysis3.0 software was used for data acquisition and pretreatment, the average serum protein profiles were obtained and compared respectively in M/Z 800 ~ 10000 range, 96 protein peaks were screened from lung squamous carcinoma group and the positive control group comparison (Figure 1); 99 differential protein peaks were screened from lung squamous cell carcinoma and benign group in the control group (Figure 2).
Figure 1 Comparison of mean serum protein profiles between lung squamous cell carcinoma (red curve) and normal control (green curve)
Figure 2 Comparison of mean serum protein profiles between lung squamous cell carcinoma group (red curve) and control group (green curve)
2: screening of protein differences of lung squamous cell carcinoma group and normal control group mean serum protein profiles compared to data obtained were analyzed by Wilcoxon test, the difference was statistically significant (PWKW< 0.01) a total of 32 protein peaks (part of the protein peak data see Table 1). The 2 protein peaks (M/Z and) with the most significant difference were analyzed. The results showed that the relative abundance of the 2 proteins in the serum protein profiles of lung squamous cell carcinoma group was much less than that of the normal control group. Using 4054.13 as the X axis and the axis as the Y axis, the results showed that the two groups had good ability to distinguish the lung squamous cell carcinoma and the normal one. Similarly, in lung squamous cell carcinoma and benign control group mean serum protein profiles compared to data obtained were analyzed for M/Z protein 2 and 4054.02 of the most significant differences between the 5065.27, their relative abundance in lung squamous cell carcinoma group mean serum protein profiles in most less than the positive control group. With 5065.27 as the X axis, the Y axis for the establishment of the coordinates of the sample distribution map, the results show that the ability to distinguish between the two lung squamous cell carcinoma and benign lung disease is good in the.
Table 1 Parameters of the first 15 peaks in the serum protein profiles of lung squamous cell carcinoma group and normal control group
Four thousand and fifty-four point one three
Four thousand two hundred and sixty-seven point four six
Four thousand two hundred and nine point seven eight
Three thousand nine hundred and fifty-four point seven eight
Four thousand one hundred and seventy-two point four three
Six thousand three hundred and thirty-two point seven one
One thousand eight hundred and sixty-five point eight two
Four thousand and ninety point nine five
Four thousand and seventy-two point six zero
One thousand seven hundred and seventy-eight point seven four
Three thousand nine hundred and thirty-five point four four
One thousand eight hundred and eighty-five point three five
Six thousand three hundred and seventy-nine point six one
Two thousand one hundred and four point eight eight
Four thousand nine hundred and sixty-three point nine one
One hundred and four point nine five
Fifty-two point eight eight
Five hundred and fifty-four point seven five
Ninety-one point eight seven
Sixty-four point four eight
Eleven point two two
Four hundred and eight point three nine
One hundred and twenty-four point two six
Nineteen point three four
Two hundred and thirty-three point four three
Fifteen point zero six
Twenty-one point one seven
Seventeen point one nine
Thirty-three point six one
Zero point zero one eight Zero Zero Zero
Zero Point Zero Zero Five Seven Zero Zero
Zero Point Zero Zero Zero two seven six
Zero Point Zero Zero Zero Five two zero
Zero point zero two four seven Zero Zero
Zero point zero one one seven Zero Zero
Zero point zero zero one nine nine zero
Zero point one four nine Zero Zero Zero
Zero point one six nine Zero Zero Zero
Thirty-eight point five one
Twenty-one point eight six
One hundred and eighty-three Point Zero Five
Thirty-three point one zero
Thirty point six eight
Ten point zero six
Four hundred and thirty-two point six nine
Seventy-four point five one
Twelve point eight four
Two hundred and fifty-three point eight seven
Twenty-one point zero seven
Thirty-seven point six three
Seventeen Point nine nine
Sixteen point seven four
Thirty-six point four eight
One hundred and forty-three point four six
Seventy-four point seven three
Seven hundred and thirty-seven point eight zero
One hundred and twenty-four point nine seven
Ninety-five point one six
Twenty-one point two eight
Twenty-four point three zero
One hundred and ninety-eight point seven seven
Thirty-two point one eight
Twenty point four four
Thirty-six point one three
Sixteen point four six
Thirty-five point one seven
Thirty-three point seven four
Seventy point zero nine
Twenty-two point five one
Sixteen point zero two
One hundred and eighty-three point four two
Eighteen point two one
Five point two four
Four hundred and thirty-one point one seven
Fifty-four point one one
Six point seven four
Two hundred and fifty-nine point nine eight
Twelve point three nine
Nineteen point five nine
Eleven point six seven
Eleven point six zero
Fifty-eight point zero seven
Fifty-six point zero four
Thirty-one point seven five
Two hundred and thirty-seven point three two
One hundred and two point seven seven
Thirty-three point two zero
Six point two two
Sixty-four point four six
Sixty-two point six four
Eleven point six nine
Fifty-one point zero seven
Thirteen point nine one
Three point eight nine
Ten point three five
Nine point four six
Forty-one point six three
Note Index: protein peaks in the spectra of the Mass: serial number; mass number; Dave: mean maximum and minimum difference; PTTA: 22 t test P; PWKW:Wilcoxon test PAD:Anderson-Darling test P value; P value; the average AveN: class N peak area or intensity; stdDevN: class N peak area or intensity standard deviation
3 screening and identification of marker proteins: a genetic algorithm was used to screen out the candidate proteins with the best sensitivity and specificity (M/Z = 1778 and 1865, respectively) (). The amino acid sequence of LC-MS/MS was identified as SKITHRIHWESASLL and SSKITHRIHWESASLL (Figure 3). Using BioworksBrowser 3.3.1 SP1 data analysis software in IPI Human (3.45) database for Sequest™ retrieval, the results show that these 2 proteins are complement C3 fragments or C3 precursors.
Figure 3 lung squamous cell carcinoma M/Z protein identification of figure 1773 and 1865 (the scarlet letter identification results within a peptide sequence)
During the process of tumor development, the secreted proteins, tumor antigens and autoantibodies can be recycled into the blood. Proteomics technology provides a new technical platform for exploring the relationship between serum and disease. With surface enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS) compared to the CLINPROT system of spherical nano magnetic beads more surface area, capture protein more, more sensitive to , especially the capture of low abundance protein or polypeptide species more, only 1 drops of serum (10 ~ 50 L), with a relative molecular mass for the 800 to 15000 range, can detect more than and 400 different peaks, to ensure the specificity of the system; it has good compatibility with mass spectrometry, direct identification of high abundance protein or polypeptide .
This study adopts CLINPROT system to analyze the serum protein in patients with lung squamous cell carcinoma and benign lung diseases and normal human expression, the selected M/Z is 2 the difference between 5065.27 and 4054.02 protein peak is the most significant, the ability to establish with the 2 kinds of different protein coordinates has good distinguish lung squamous cell carcinoma and benign lung the latter disease; screening out M/Z 2 between the 4054.13 and 4267.46 protein peak is the most significant, also has good ability to distinguish lung squamous cell carcinoma and normal people. Freed et al. CLINPROT was used to study the expression of M/Z 5064 differential protein in squamous cell carcinoma of the head and neck and normal human serum in . In this study, the differentially expressed proteins of M/Z 5065.27 may be the same protein.
In this study, the differential protein peaks of M/Z 1778 and 1865 were identified. The results showed that both of them may be the same source as complement C3 fragment or C3 precursor. Complement C3 precursor is a kind of double chain protein, which is linked by two disulfide, and can be activated into C3 by many ways. Some scholars have studied the serum of patients with multiple tumor metastasis after operation, and found that the complement is in a state of inactivation. De Visser et al. Found that the independent complement pathway plays an important role in the aggregation of  cells and the occurrence of tumor. In the 25 known species of soluble complement protein, complement C3 is currently one of the most identification function, it can be through the change of structure and at least 25 different protein interactions, is a regulator of infiltrating inflammatory cells. Mizuno and other  studies have shown that the secondary complement C3 loss is much more than congenital deficiency. Okroj  found that soluble complement non small cell lung cancer cells can produce more protein binding protein factor I and C4b regulation, increase their combined with C3b, C4b, generated fragment of complement activity reduced, thereby reducing the number of C3, reduce the activity of the cells of the host immune function decline, eventually leading to immune the escape of tumor cells. Villanueva and Freed, such as  and , were used to identify the complement C3 fragment (M/Z 4) in the solid tumors of head and neck squamous cell carcinoma (CLINPROT = 1865).
In this experiment the protein peaks of WCX beads can capture more stable, but it is detected with cationic groups of proteins in serum, serum protein is not all. Therefore, try other types of chips or a combination of multiple chips, can improve the effect of screening differential proteins, may find more and more valuable tumor marker protein.
In summary, there are serum proteins between patients with lung squamous cell carcinoma and benign lung diseases and normal expression, the application of CLINPROT system to the screening of protein markers may have better diagnostic value for lung squamous cell carcinoma. Of course, it is also necessary to expand the sample size for further study.
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(Editor: Yan Ming)