CN101776642B

CN101776642B - Mass spectrometric detection method for ca-dependent secretory protein 1 in human serum

Info

Publication number: CN101776642B
Application number: CN 201010105552
Authority: CN
Inventors: 沈锡中; 薛如意; 刘韬韬; 董玲
Original assignee: Zhongshan Hospital Fudan University
Current assignee: Zhongshan Hospital Fudan University
Priority date: 2010-02-04
Filing date: 2010-02-04
Publication date: 2012-10-31
Anticipated expiration: 2030-02-04
Also published as: CN101776642A

Abstract

The invention relates to a mass spectrometric detection method for ca-dependent secretory protein 1 in human serum, which is characterized in that the mass spectrometric detection method comprises the following steps: taking human serum and using magnetic bead attachment protein for obtaining magnetic bead enrichment liquid; adding the magnetic bead enrichment liquid into a matrix solution; carrying out the matrix-assisted laser dissociation ionization flight time mass spectrometric detection according to the following conditions: a first ion source voltage: 20kV; a second ion source voltage:18.6kV; crystal voltage: 7.6kV; pulse ion time: 320ns; polarity: positive; matrix inhibition mode: gate control; gate control strength: maximum: suppression high limit: 600Da; detector: 1400-1500 V; sample rate: 1.00Gs/s; electrical acquisition: enhancing, 100mV; real-time fluency: high; laser frequency: 20Hz; laser attenuation: closing 73% and ranging 20%; mass to charge ratio is 5336 plus or minus 1; and the peak flowing is the ca-dependent secretory protein 1. The method has the advantages of short time and high sensitivity.

Description

The Mass Spectrometer Method method of ca-dependent secretory protein 1 in a kind of human serum

Technical field

The present invention relates to the Mass Spectrometer Method method of ca-dependent secretory protein 1 in a kind of human serum; Be specifically related to a kind of employing enrichment with magnetic bead associating ground substance assistant laser parsing/ionization time of flight mass spectrometry (matrix-assisted laserdesorption/ionisation time-of-flight mass spectrometry; MALDI-TOF MS) method of screening ca-dependent secretory protein 1 (caps1) from human serum belongs to the method for protein detection technical field.

Background technology

(Calcium-dependent secretion activator 1 CAPS-1) is a kind of calbindin to ca-dependent secretory protein 1, can promote neurotransmitter, neuropeptide vesica to be transported to cell membrane and further discharge secretory granules.The main detection method of caps1 comprises protein immunization marking method, enzyme linked immunosorbent assay at present, and their shortcoming is consuming time, effort, and insufficient sensitivity is high, is inappropriate for the screening of extensive sample.

Summary of the invention

The objective of the invention is to overcome the problems referred to above, the Mass Spectrometer Method method of ca-dependent secretory protein 1 in a kind of human serum is provided.

In order to achieve the above object, technical scheme of the present invention provides the Mass Spectrometer Method method of ca-dependent secretory protein 1 in a kind of human serum, it is characterized in that, concrete steps are:

The first step: take people's whole blood, solidify 1 hour under 4 degrees centigrade, centrifugal; Get upper serum, be kept in-80 degrees centigrade of refrigerators, weak cation is exchanged magnetic bead or the affine copper ion magnetic bead of immune metal combine liquid in thin-walled PCR pipe, to mix with magnetic bead; Add serum sample, incubated at room 5min places thin-walled PCR pipe and carries out magnetic bead in the magnetic bead separation vessel and separate; Remove supernatant, after last magnetic bead is washed with the magnetic bead cleansing solution, add the magnetic bead eluent; Thin-walled PCR pipe placed carry out magnetic bead in the magnetic bead separation vessel and separate, obtain eluent, eluent is mixed obtaining enrichment with magnetic bead liquid with magnetic bead stabilizing solution;

Second step: the enrichment with magnetic bead liquid that the first step is obtained adds matrix solution, carries out the ground substance assistant laser ionization time of flight mass spectrometry that dissociates by following condition and detects: first ion gun voltage: the 20kv; Second ion gun voltage: the 18.6kv; Crystal voltage: 7.6kv; The pulse ion time: 320ns; Polarity: just; Matrix suppression mode: gate; Gate intensity: maximum; Suppress high limit: 600Da; Detecting device: 1400-1500v; Sample rate: 1.00Gs/s; Electronics obtains: strengthen 100mv; Smooth in real time: height; Laser frequency: 20Hz; Laser attenuation: close 73%, scope 20%; The peak examination criteria: signal to noise ratio (S/N ratio) is greater than 5, and the 2Da peak width is filtered, at most peak number 200;

The 3rd step: analyze the mass spectrogram that second step obtained, observe mass-to-charge ratio is whether 5336 ± 1 places have the peak in the mass spectrogram,, then contain ca-dependent secretory protein 1 in the human serum in the serum if the peak is arranged.

Advantage of the present invention is: this method is selected for use fast and the responsive ground substance assistant laser ionization time of flight mass spectrometry that dissociates detects serum caps1; Belong to the proteomics research category; Ground substance assistant laser desorption ionization flight time mass spectrum is a very effective instrument finding and identifying aspect albumen or the polypeptide of disease association; Be used to seek multiple disease specific sign, the method for this technical tie-up magnetic particle enrichment haemocyanin more fast, effectively and accurately.This is that hypersensitivity and the repeatable technology on basis is allowed to condition at the protein science research field and takes the course of its own with the magnetic particle.The each detection of this method only needs tens of minutes time; The enrichment and the detection of serum caps1 albumen have been accomplished; Simple to operate with respect to traditional protein immunization marking method, enzyme linked immunosorbent assay (consuming time more than 8 hours), save time, be convenient to the detection of sample in enormous quantities; Because of not relying on many restrictions that biochemical reaction does not receive reaction conditions, repeatability is high; The detection sensitivity of classic method is a Gamma Magnitude in addition, and the detection sensitivity Da Nake level of this method is significantly higher than traditional method.

Description of drawings

Fig. 1 is a through the human serum Mass Spectrometer Method figure behind the enrichment with magnetic bead.

Embodiment

Specify the present invention below in conjunction with embodiment.

Embodiment

1. main agents:

Weak cation exchange magnetic bead MB-WCX kit (Bruker Daltonik GmbH, Germany),

Immunity metal affine copper ion magnetic bead MB-IMAC Cu kit (Bruker Daltonik GmbH, Germany)

Alpha-cyano-4-hydroxycinnamic acid α-Cyano-4-hydroxycinnamic acid (HCCA) (sigma, the U.S.)

Acetone (sigma, the U.S.)

Ethanol (sigma, the U.S.)

AnchorChip ^TMTarget (Bruker Daltonik GmbH, Germany)

2. consumptive material and instrument:

Vacuum test tube (BD Vacutainer ^TM, the U.S.)

Low temperature refrigerator (SANYO, Japan)

Vortex mixed appearance (H101, last Haikang standing grain photoelectric instrument company limited)

Constant temperature supercentrifuge (Z400K, HERMLE, Germany)

Magnetic bead separation vessel (Bruker Daltonik GmbH, Germany)

Autoflex II ground substance assistant laser dissociating ions time-of-flight mass spectrometry (Bruker Daltonik GmbH, Germany)

LC/MS/MS combined instrument (u.s.a. applied biosystem company, Shanghai)

Other: syringe, Eppendorf pipe, application of sample rifle, rifle are first-class

3. the collection of biological sample:

Select BD Vacutainer for use ^TMAfter vacuum test tube (yellow skull) is taked people's whole blood, solidified 1 hour under 4 degree, press 3000rpm 10min altogether, get upper serum, be kept in the Eppendorf pipe for use in-80 degree refrigerators with hydro-extractor.

4. serum sample pre-treatment:

(1) on the vortex mixed appearance, weak cation is exchanged MB-WCX magnetic bead solution or the thorough mixing 1min of the affine copper ion MB-IMACCu of immune metal magnetic bead solution.

(2) 10 μ l MB-WCX magnetic beads combine liquid and 10 μ l WCX magnetic bead solution to be transferred in the thin-walled PCR pipe of a standard, up and down the pressure-vaccum mixing.(or with 50 μ l MB-IMAC Cu magnetic beads combination liquid pre-service, 5 μ l MB-IMAC Cu magnetic bead solution; Thin-walled PCR pipe is put into the magnetic bead separation vessel; Thin-walled PCR pipe is moved 10 times between adjacent holes, assemble magnetic bead, carefully remove supernatant with rifle in tube wall 20s; Repeat secondary, MB-IMAC Cu magnetic bead is resuspended in 20 μ l MB-IMAC Cu magnetic beads combines in the liquid).

Add 5 μ l human serum samples, pressure-vaccum is 5 times up and down, mixing liquid.Incubated at room 5min.PCR pipe is placed in the magnetic bead separation vessel, pipe is moved forward and backward 3 times after, assemble magnetic bead in tube wall 1min, carefully remove supernatant with rifle.

Xiang Guanzhong adds 100 μ l magnetic bead cleansing solutions, before and after the PCR pipe is in the magnetic bead separation vessel, moves around 10 times, notes moving of magnetic bead.Assemble magnetic bead 20s on tube wall, carefully remove supernatant with rifle.Repeat this step twice.

Add 5 μ l MB-WCX magnetic bead eluents (or 10 μ l MB-IMAC Cu magnetic bead eluents), 10 times magnetic bead is dissolved from tube wall, wait for 5min through strong pressure-vaccum up and down.

Pipe is put into the magnetic bead separation vessel, on tube wall, assemble magnetic bead 2min, the supernatant that will contain the wash-out molecule shifts in the clean pipe.In eluent, add 4 μ l magnetic bead stabilizing solution, the strong mixings of pressure-vaccum up and down.

The sample of 1 μ l wash-out is added the matrix solution (HCCA solution, it is at ethanol: acetone is that concentration is 0.3g/l in 2: 1 the solution) of 10 μ l, put 1 μ l to MALDI-TOF mass spectrum target.

5. mass spectrum condition:

First ion gun voltage: the 20kv; Second ion gun voltage: the 18.6kv; Crystal voltage: 7.6kv; The pulse ion time: 320ns; Polarity: just; Matrix suppression mode: gate; Gate intensity: maximum; Suppress high limit: 600Da; Detecting device: 1400-1500v; Sample rate: 1.00Gs/s; Electronics obtains: strengthen 100mv; Smooth in real time: height; Laser frequency: 20Hz; Laser attenuation: close 73%, scope 20%.The peak examination criteria: signal to noise ratio (S/N ratio) is greater than 5, and the 2Da peak width is filtered, at most peak number 200.

6. testing result:

As shown in Figure 1, the human serum Mass Spectrometer Method figure for behind the portion process enrichment with magnetic bead, analyze as follows:

Sequence number	Mass-to-charge ratio	Peak area
			1	1206.852	233.2806

2	1234.907	164.5131
			3	1308.592	366.7388
4	1316.401	519.6103
			5	1321.026	344.6194
6	1329.471	1089.298
			7	1338.959	435.7691
8	1349.922	221.7832
			9	1364.439	212.3075
10	1385.022	9.148874
			11	1430.335	294.3772
12	1465.247	379.206
			13	1471.953	310.7988
14	1545.199	78.50821
			15	1619.369	1522.463
16	1631.186	2146.741
			17	1717.474	396.6772
18	1725.901	103.1431
			19	1738.477	138.6453
20	1746.532	684.2241
			21	1763.722	960.641
22	1777.051	548.2078
			23	1808.028	262.3039
24	1813.866	498.4405
			25	1829.415	381.9238
26	1867.389	236.5915
			27	1888.154	3469.796
28	1945.111	7614.77
			29	1979.032	1031.452
30	2046.854	665.103
			31	2081.432	7881.219
32	2091.556	1892.181
			33	2104.803	10728.67
34	2209.504	3756.636
			35	2294.414	2939.391
36	2344.978	1147.049
			37	2353.17	103.2464
38	2364.338	1158.016
			39	2404.817	773.1838
40	2420.323	1115.697
			41	2481.959	731.6669
42	2559.922	9919.333
			43	2611.364	190.0135
44	2623.927	259.4034
			45	2630.597	365.6349

46	2645.672	4443.032
			47	2660.64	77700.35
48	2725.39	1544.949
			49	2739.036	941.1075
50	2769.317	14584.53
			51	2862.939	31985.49
52	2883.266	9414.285
			53	2902.075	3810.259
54	2932.038	24462.84
			55	2951.82	41541.18
56	3192.049	21752.23
			57	3222.155	2963.911
58	3224.215	2963.911
			59	3241.183	46399.18
60	3263.096	40800.38
			61	3447.513	603.8208
62	3505.064	1879.339
			63	3523.206	4182.684
64	3538.167	1750.341
			65	3586.819	664.058
66	3616.573	126.8505
			67	3636.45	1764.688
68	3678.28	2093.34
			69	3680.634	2093.34
70	3697.631	795.8046
			71	3726.096	1776.15
72	3952.973	8315.439
			73	4054.517	11959.5
74	4073.242	4533.026
			75	4090.748	56336.94
76	4121.616	2703.324
			77	4154.886	1807.75
78	4168.399	3301.79
			79	4209.553	240623.3
80	4269.454	10226.33
			81	4526.278	1213.465
82	4643.493	67880.99
			83	4672.354	14847.94
84	4787.175	5741.917
			85	4915.494	1385.752
86	4936.063	834.1923
			87	4963.46	41928.48
88	5065.108	8760.362
			89	5290.733	806.0496

90	5336.44	130785.5
			91	5398.947	1636.666
92	5479.533	504.1858
			93	5523.46	4029.33
94	5904.121	401500.5
			95	5966.6	65.17127
96	6089.927	2243.643
			97	6630.462	12355.21
98	7765.259	36881.82
			99	9288.768	196286.3
100	9347.858	5288.739

Mass-to-charge ratio is the peak that the peak at 5336.44 places is ca-dependent secretory protein 1 in the last table.

7, utilize the liquid chromatography mass GC-MS further to identify:

Enrichment with magnetic bead liquid is identified with the liquid chromatography mass GC-MS, with the MASCOT program mass spectrometric data is carried out online international albumen parameter (International Protein Index, IPI) database (http://www.ebi.ac.uk) inquiry.

Wherein, liquid chromatography-mass spectrography/mass spectroscopy device is connected the LTQ-Orbitrap mass spectrum and is constituted by the nanometer stream liquid chromatography pump of two LC-20AD, SIL-20AC automatic sampler, a LC-20AB miniflow liquid chromatography pump.Sample fills in the CAPTRAP post; Flow velocity 50ul/ branch gets into the C18 reversed-phase column and carries out liquid phase separation, flow velocity 500nl/ branch after 3 minutes; Moving phase is 5% acetonitrile and 0.1% formic acid (A mutually, filling phase), 95% acetonitrile and 0.1% formic acid (B mutually), B phase linear gradient 5%-45% (60 minutes).Sample after the separation gets into mass spectrometer through ADVANCE30um silicon dioxide interface end, spray voltage 1kv, and 180 ℃ of temperature, data rely on pattern, each complete scanning of the mass spectrum mass range 400-2000Da, resolving power 100000.The peptide section is at LTQ certain applications helium collision induced dissociation, and mark is dissolved from energy value 35%.The peak that dissociates of band 2+ and 3+ is selected does mass spectrum/mass spectrophotometry.Select for use BioWorks 3.3.1 sp1 software that IPI human protein database is carried out on-line search, condition: 2 possible error sites, the polypeptide mass deviation allows for 10ppm, and the fragment mass deviation allows for 1.00Da.Mass-to-charge ratio is that 5336 ± 1 material peak is accredited as caps1 albumen.

Claims

1. the Mass Spectrometer Method method of ca-dependent secretory protein 1 in the human serum is characterized in that concrete steps are:

The 3rd step: analyze the mass spectrogram that second step obtained, observe mass-to-charge ratio is whether 5336 ± 1 places have the peak in the mass spectrogram,, then contain ca-dependent secretory protein 1 in the serum if the peak is arranged.