CA1339251C - Derivatized glass supports for peptide and protein sequencing - Google Patents

Abstract

ABSTRACT
Peptides or proteins are sequenced by stepwise degradation while immobilized on a glass support derivatized with a silica-binding substance bearing a free acid group, especially a sulfonic acid group. The support is preferably derivatized with 2-(4-chlorosulfonyl phenyl) ethyl trimethoxysilane. Peptide sequencing performance is improved if the support is also derivatized with a monomeric silica-binding substance bearing a free quaternary ammonium group, such as n-trimethyoxysilyl propyl-N,N,N-trimethyl ammonium chloride.

Description

13392~1 DERIVATIZED GLASS SUPPORTS FOR ~ll~E
AND PROTEIN SEOUENCING

BACKGROUND OF THE INVENTION
This lnventlon relates to the use of a glass support derlvatlzed wlth 2-(4-chlorosulfonylphenyl) ethyl trlmethoxy sllane (sllyl CSP), N-trlmethoxysllylpropyl-N,N,N-trlmethyl ammonlum chlorlde (sllyl TMA) or a comblnatlon thereof, ln the sequencing of peptldes or protelns.
The prlmary sequence of amlno aclds in a peptide or protein is commonly determined by a stepwise chemical degradation process ln which amino acids are removed one-by-one from one end of the peptlde, and identlfled. In the Edman degradation, the N-terminal amlno acld of the peptlde ls coupled to phenylisothiocyanate to form the phenylthiocarbamyl (PTC) derivatlve of the peptlde. The PTC peptlde ls then treated with strong acid, cyclizing the PTC peptide at the first peptide bond and releasing the N-terminal amino acld as the anlllnothlozollnone (ATZ) derlvatlve. The ATZ amlno acid, which is highly unstable, is extracted and converted into the more stable phenylthiohydantoin (PTH) derivative and identified by chromatography. The residual peptide ls then subiected to further stepwlse degradatlon.
Automatlc protein sequencers ln which the Edman degradatlon reactlons are carried out in a film on the inside surface of splnnlng cup are known. Edman and Begg, Eur. J.
Biochem., 1:80 (1967); Penhast, U.S. 3,725,010. A quaternary ammonlum salt, 1,5-dlmethyl-1,5-dlazundecamethylene polymethobromlde (Polybrene) has been used as a sample X

2 ' 13392Sl carrier.
Schroeder, Meth. Enzymol., 11:445 (1967) and Jentsch, Jr., Proc. Flrst Int'l Conf. on Meth. in Proteln Sequence Anal. 193 (1975) modified the liquid phase Edman degradation by first non-chemically depositing the protein or peptide onto a paper strip.
Laursen, Meth. Enzymnol., 25 344 (1972) covalently attached peptides to an insoluble resin prior to sequencing.
Wachter, et al., FEBS Lett., 35 97 (1973) immobilized the peptides on controlled pore glass beads derivatized with 3-aminopropyltriethoxysilane. The resulting aminopropyl glass binds to free carboxyl groups of the peptides. Dreyer, U.S.
4,065,412 favours immobilizing the peptides, either by covalent linkage or by adsorptlon, onto a macroporous reaction support surface of polystyrene or glass.
Hood, U.S. 4,704,256 and 4,603,114 teaches embedding the sample in a permeable solid matrix formed as a thin film on a support such as a glass fiber sheet. The matrix is preferably a polymeric quaternary ammonium salt, since the positively charged quaternary ammonium groups interact strongly with the negatively charged glass surface.
Figures 17A, 17B and 17C in the Hood patents illustrate three approaches to improving sample retention by immobilizing the protein or peptide. Figure 17A shows covalent attachment to derivatized glass, as in Wachter.
Figure 17B shows physical adsorption of the sample to the support, as in Schroeder. Figure 17C illustrates embedding 3 1~39251 the sample in a matrlx coverlng the glass, as ln Hood.
Saunders, U.S. 3,987,058 dlscloses the use of sulfonated aralkyl slllcas as catlon exchangers. The sodium salt of sulfobenzylsilica was used to separate nitrosoproline from prollne and other components of cured meat samples.
There ls no discussion of protein sequencing.
Glaich, U.S. 4,705,725 relates particularly to support structures comprlsing slllca to whlch is covalently attached a monofunctlonal sallne containlng at least two sterically protectlng groups attached to the sllicon atom of the sllane. For use in peptlde sequenclng, thls sllane ls substituted with a quaternary amlno group so as to interact with peptides in a manner slmllar to that of Polybrene. For use ln catlon exchange chromatography, this sllane ls substltuted wlth a -~CH2)3-C6H4-SO3H group.
Sllanes have been used to couple antlgens or antibodies to glass. Weetall, U.S. 3,652,761.
SUMMARY OF THE INVENTION
Present methods of gas-phase or so-called pulsed-llquld proteln sequenclng rely on embedding the sample ln amatrix (Hood) or covalent attachment of the sample to a support (Laursen). This is to prevent dislodglng the sample from the support during liquid solvent washes and transfers.
Covalent attachment has the advantage of permanently locatlng the sample ln the reactlon chamber wlthout danger of dlslodglng lt durlng llquld-phase chemlstry or washlng. The dlsadvantages are extra chemlcal reactions to attach the sample to the reaction support, inefficiencies inherent in these attachment procedures and low yields of residues in the sample which are involved in the attachment to the support.
Sample embedding has the advantage of being a more or less universal immobilization method without the extra sample manipulation associated with covalent attachment. Ma~or disadvantages include high levels of contaminating artifacts on analytical systems used for PTH amino acid identlfication and the necessity to run the sequencer for several cycles before the actual sample is loaded. Also, it is common practice to add amines which act as scavengers of these amino-reactive contaminants which would otherwise react with the N-terminal amino group of the sample and block it to Edman degradation. Since many of these contaminants show up as artifacts in PTH analysis, the sequencer must be run for several cycles to lower the level of these artifacts before the sample is loaded. This "precycling," as it has come to be known, uses expensive chemicals and valuable time. Another dlsadvantage of carrier methods relates to the Edman chemicals and washing solvents having to diffuse into and out of the matrix. Since the sample is trapped within a matrix and not fully exposed on the surface of the support, reaction and washing efficiency may be compromised.
In accordance with the present invention there is provided a method of sequencing a peptide or protein which comprises immobilizing the peptide or protein on a support and sequencing the peptide or protein by stepwise degradation, the 1~392~

improvement whlch comprlses provlding a glass support derlvatized with a silica-binding substance having a free acid group, said acid having a pKacl, said derivatized support being capable of retaining said peptide or protein.
The present invention also provides a method of sequencing a peptide or protein which comprises immobilizing the peptide or protein on a support and sequencing the peptide or proteln by stepwise degradation, the improvement which comprises provlding a glass support derlvatized wlth a substance which covalently binds to silica, said substance having a quaternary ammonium group, said derivatized support being capable of retaining the peptide or protein.
The present invention further provides a method of sequencing a peptide or protein which comprises immobilizing the peptide or protein on a support and sequencing the peptide or protein by stepwise degradation, the improvement which comprises immobilizing the peptide or protein on a glass support derivatized with a first silica-binding substance havlng a free acid group, sald acld group having a pKa<l, and a second sllica-blnding substance having a free quaternary ammonlum group, wherein sald support is capable of retalning a peptide or protein.

The present invention yet also provides a derivatized glass support comprising a sillcaceous support derivatized with a first silica-binding substance having a free acid group, said acid group having a pKa~l, and a second silica-binding substance having a free quaternary ammonium group, said support being capable of retaining a peptide or protein.
We have found that certain derivatized glass supports bind peptides and proteins well enough to minimize loss of the sample during solvent and reagent delivery, while still releasing the amino acid derivatives as they are cleaved during the degradation process. These are "silyl CSP," 2-(4-chlorosulfonyl phenyl~ ethyl trimethoxysilane, and "silyl TMA," N-trimethoxysilyl propyl-N,N,N-trimethyl ammonium chloride.
Both the protein and peptide supports are made by chemical modification of the surface of the glass itself.
This derivatization is a covalent process and results in the permanent alteration of the glass fiber surface. The silyl TMA and silyl CSP are bifunctional reagents. The reagents are attached to the glass through the silyl groups allowing the other functional group free to interact with the protein or peptide. Since the reagent ls covalently attached and since it is in a monolayer on the surface of the glass, it cannot form a matrix which envelopes the sample. The sample must interact with the free functional groups through a comblnation of electrostatic and hydrophobic forces. This type of interaction is very desirable because it allows the sample to interact with reactants directly, unencumbered by a matrlx through which chemicals must diffuse.
While it is possible to make one type of derlvatlzed glass support which will work for both short peptides and 133~251 protelns, we have found lt advantageous to customlze the surface for either small or large molecules. Glass derlvatized wlth only sllyl-CSP works very well as an anchor for small molecules (peptldes with molecular welghts under 8000 daltons). Larger peptldes and protelns also adhere well to thls surface but PTH amlno aclds wlth posltlvely charged slde chalns are not efflclently extracted from the support and give very low ylelds upon subsequent analysls. If the glass surface ls treated flrst wlth sllyl-CSP and then sllyl-TMA, this effect ls greatly reduced without affectlng the ablllty of the surface to retaln the sample. "Problem" peptldes may be sequenced by adiustlng the ratios of the two silanes attached to the glass surface. Pure silyl-TMA derivatlzed glass works well as a support for large molecules Ipeptides and protelns wlth molecular weights over 8000 daltonsl, but ls less satlsfactory at retainlng smaller peptldes on the support surfaces.
Although the speclflc chemicals mentioned above for sllyl derivatlzed supports are used ln the preferred embodiment, modified derlvatlves containing the same or slmilar functlonal groups may work as well or better. For example, any approprlate acld group wlth a sufflciently low pKa (~1) may be substltuted for the sulfonlc acld group ln the preferred embodlment. Examples of other aclds whlch fall into thls category lnclude but are not llmlted to phosphorlc, plcrlc, hydrochloric, trifluoroacetic, trichloracetic, chromic, hydroiodlc, pyrophosphorlc, among others. We have found that the presence of the acidlc group ls critical to the performance of the peptide support. Omitting the acid group and uslng a compound such as phenethyltrlmethoxy silane results in a support which does not retain peptides, independent of the percentage of chemical used in the derivatization. If a compound such as 3-(trimethylsilyl)-1-propane sulfonic acld is used, a compound which lacks a phenyl ring but has an acid group, the resulting derivatized surface retalns peptides. Evidently, it is the presence of the acld group and not the phenyl ring or carbon chain which promotes sample retention. However, since this compound is a solid at room temperature~ it is much less convenient to use than the silyl-CSP.
Similarly, the silyl-TMA may be replaced by another quaternary ammonium group bearing compound which covalently binds to glass.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 compares the yield of PTH-proline in the first cycle of analysis of a decapeptide using a gas-phase sequencer with (a) a support treated with Polybrene or (b) a silyl-CSP~TMA derivatized support.
Figure 2 compares the yield of PTH-histidine in cycle 2 of the analysis of the same decapeptide, using a gas-phase sequencer with (a) a Polybre~e-treated support, (b) a silyl-CSP/TMA derivatlzed support, and (c) a silyl-CSP/TMA
derlvatized support with reduced CSP relative to support ~b).
Figure 3 shows the first 15 cycles of a gas-phase 13392~1 sequencer run of 8 picomoles of beta-lactoglobulin A using the silyl-TMA support.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is thus directed, in one embodiment, to the use of a silica-binding substance having a poly (amino) acid-reactive group to prepare a support suitable for sequencing peptides and/or proteins. The silica-binding substance is preferably an organosllane, having the formula RnSiXI~n!, where n is 1 to 3 and X is the group which reacts with the inorganic substance. X is a hydrolyzable group, typically, alkoxy, acyloxy, amlne or chlorine. The most common alkoxy groups are methoxy and ethoxy. R is a nonhydrolyzable organic radical that possesses a functionality that interacts with ~mino acids.
The preferred concentration is 0.25-2% ~v/v) of silica-blnding substance in the solvent after dilution.
The support is preferably derivatized with an anionic silane such as the hydrolyzed form of silyl-CSP, or with both an anionic silane and a cationic silane such as silyl-TMA.
The chlorosulphonyl group on the silyl-CSP compound is known to react with primary and secondary amines. Thus, using this active group in the presence of peptides or proteins will block them to sequence analysis by the Edman degradation, which requlres a free amino group for the coupling reaction. To overcome this problem, the chlorosulphonyl group is quantitatively converted to the sulfonic acld derivative. This hydrolysis reaction conveniently occurs during the procedure for attaching the sllyl-CSP to the glass surface.
The glass surface then has negatively charged sulfonlc acld groups wlth a very low pKa (~1). Thls greatly facilitates the blndlng of posltlvely charged molecules to the glass. However, lt ls observed that peptldes wlth a net negative charge and those whlch are neutral in charge also bind well and are not removed from the surface during the sequenclng procedure. From experlments conducted with other types of derivatlzed glass supports, lt appears that the peptlde is most likely to be removed from the surface during the ATZ solvent extractlon followlng the acid cleavage step of the Edman degradation. At this point, the peptlde will have a net positlve charge, no matter what lts composltlon may be, due to the fully protonated N-termlnal amlno group. If the pKa of the surface groups ls sufficlently low, the surface will still have a net negatlve charge promoting electrostatic interaction with the peptide. The ATZ amino acid derivative is a small uncharged hydrophobic molecule under these conditions and will be easlly extracted wlth solvent away from the charged peptlde. The only exceptions will be ATZ
histldlne and ATZ arglnlne whlch wlll have a net posltive charge and thus be more difflcult to extract. In fact, lf the surface of the glass has too hlgh a concentratlon of sulfonlc acid groups, ATZ His and Arg will be quantitatively retained on the support during extraction.

11 1333~51 Lysine, which may be posltively charged under acidlc conditions, will not be charged after the first Edman coupling reaction since the epsilon-amlno group reacts wlth PITC to form the epsilon-PTC derivative maklng ATZ lyslne very hydrophoblc and easlly extracted.
During the coupling reaction, a baslc envlronment maintalns a net negatlve charge on the support. The peptlde will also be negatively charged via the carbonyl group since the amlno termlnus is involved in the neutral PTC group.
During extraction of coupling reaction by-products the peptide remains on the support even though the electrostatic environment would appear to be unfavorable.
Evidently, a number of effects are combining to form a very strong attractive force. Perhaps partial positive charges within the derivatlzed peptide bind it to the hlghly negative surface. This highly desirable effect is somewhat offset for very short hydrophobic peptides slnce a lower CSP
to TMA ratlo (See Figure 3) may lower thelr affinity for the support and result in poor ylelds of PTH amlno acids as the sequencing run nears the carboxyl terminus. With more hydrophilic peptides, thls ls not the case and ylelds near the C-termlnus are still good.
In the pure sllyl-CSP support, the preferred concentration of silyl CSP in the solutlon applied to the glass is 1-2%. In the hybrld sllyl-CSP/sllyl TMA support, the preferred concentration of sllyl-CSP ls 0.1 to 0.25% and the preferred concentration of silyl-TMA ls 0.5-2.0%. A pure silyl-TMA support may also be prepared.
We have found that the method used for the preparation of the glass surface is very critical to the performance of the supports in proteln sequencers. Previously publlshed methods for sllyl derivatization of glass for use in protein sequences have proven to be very unrellable. Many of these methods requlre extensive pretreatment of the surface to be derlvatized with varlous aclds (so-called "acld etchlng") and bases to make the silylizatlon successful. We have found these pretreatment methods completely unnecessary and ln most cases detrimental. In partlcular, rlnslng wlth solvent prior to heat treatment is undesirable. The following methods produce surfaces with consistently high afflnlty for protelns and peptides. These examples illustrate the preparation and use of the preferred embodlments of the present invention.
Example 1 The following example illustrates a method which yields a surface with a very high affinlty for poly(amino) acld molecules over about 8000 daltons. It allows sequence analysis on very low quantities of sample. Indeed, the detection method used in the PTH analysis is the limlting factor rather than the amount of sample sequenced. A sllyl-TMA derivatized glass surface for use as a protein support is prepared as follows:
1. Pour 95 ml of HPLC-grade methanol into a 100 ml glass graduated cylinder and add 5 ml HPLC-grade water.
2. Mix well and add 6 ml of a 50% solutlon of sllyl-TMA

~1!~. ?

,'C .' I

133!~2~1 ln methanol. (The effective concentratlon ls thus about 3% of sllyl-TMA).
3. Mix well and let stand for 5 mins., to allow hydrolysls of the silyl groups to take place - do not let stand for more than 5 mlnutes.

4. Pour the solutlon lnto glass petri dlshes.

5. Place glass fiber disks (e.g., Whatman GF/F or GF/C
glass flber disks) into the solution, being certain to wet them completely.

6. Incubate for 5 mlnutes with frequent shaking and turning as necessary to ensure good exposure of the glass to the solutlon - it is important to keep the disks completely covered with solutlon.

7. At the end of 5 minutes hang the disks in an oven at 110~C for 30 minutes. Do not rinse the disks with solvent prlor to heat treatment.

8. While the disks are in the oven, discard the solution in the petri dishes and replace it with fresh methanol.

9. At the end of the oven incubation, remove the disks and place them in the dishes containlng methanol and rlnse thoroughly by swlrllng.

10. Place each disk into a Buchner filter funnel and wash with three funnel volumes of methanol using gravity feed.

11. Place the washed disks into the vacuum chamber and dry at room temperature for at least 30 minutes.
Other glass supports may be used in place of Whatman 133~251 GF/F or GF/C filters.
The pure silyl-TMA support does not readily retain peptldes of less than 30-40 amino acids. The pure silyl-CSP
support or the hybrid support are preferred for sequencing short peptides.
Example 2 A silyl-CSP derivatized glass support is preferred for use as a peptide support if histldlne and arglnlne are not ln the peptide. This type of surface strongly retains ATZ
histidine and ATZ arginine so that they are not extracted well from the reaction support. Adding a compound such as silyl-TMA as outlined ln example 3 below greatly reduces thls effect, posslbly by competing for the negative sites present on the sllyl-CSP surface. A pure sllyl-CSP surface for use as a peptide support is prepared as ln Example 1, but addlng 2 ml of a 50% solutlon of sllyl-CSP ln methylene chlorlde. ~After step 2, the effective concentration ls 1% of silyl-CSP).
Example 3 A mixed silyl-CSP/silyl-TMA derivatized glass surface for use as a peptide or protein support may also be made, and this is the most preferred support. The mixture offers superior peptide performance as compared to pure silyl-CSP or pure silyl-TMA since PTH histidine and PTH arginine are extracted well yet surface affinity for even small hydrophobic peptides is excellent. Pure silyl-TMA does not work at all on most small peptides. The mixed support is prepared by first preparing a silyl-CSP support as in Example 2, but wlth 0.5 ml -~ 76447-1 133~2~1 of a 50% solution of silyl-CSP in methylene chloride (i.e., 0.25% after dilutlon), and then following the Method of Example l, but with 4 ml of a 50% silyl-TMA in methanol (l.e., 2% after dilution).
Example 4 Figure 1 illustrates the value of the hybrid CSP/TMA
support for peptide analysis. The decapeptide Pro-His-Pro-Phe-His-Phe-Phe-Val-Tyr-Lys (200 PM) was loaded onto an automated gas-phase sequencer (porton Instruments PI 2020).
Thls decapeptide presents a conslderable challenge to most automated gas or gas/liquid-phase sequencers because of its extremely hydrophobic C-terminal.
The sequencer was equipped with either the hybrid CSP/TMA support of Example 3, or a support treated with Polybrene. The latter support was prepared by pipetting 1.5 mg Polybrene in 15 ul H~0 onto an underivatized glass fiber support, and permitting it to evaporate. As is seen in Figure 1, the support treated with Polybrene (lA) gives a much higher background level of artifacts (peaks marked "*") than does the CSP/TMA support during the first analytical cycle.
Example 5 Figure 2 shows the result of modifying the CSP/TMA
ratio from .25% CSP/2% TMA (Fig. 2B) to .1% CSP/2% TMA (Fig.
2C). The selection of the latter ratio greatly increased the yield of PTH-histidine ln cycle 2. The silyl CSP component of the hybrld support usually should not exceed a concentration of 1%, and 0.25% is preferred. When sequencing a peptide X 76447-l 133~251 known to be rlch in Hls or Arg, use of a hybrid support prepared using an even lower silyl-CSP concentration may be deslrable.
Example 6 Flgure 3 shows the flrst 15 cycles of gas-phase sequencer run of beta-lactoglobulin A and illustrates the excellent performance of the silyl-TMA proteln support with quantlties of sample which approach the detecting limit of the analytlcal system. Only 8 picomoles of proteln were loaded onto the support yet the sequence can be easily determined by visual observation. In this experiment, 75% of the PTH amino acid delivered to the sequencer's fraction collector was analyzed. The yield of PTH leucine at cycle one is greater than 60% and the repetitive yield between PTH vallne at cycle 3 and cycle 15 is greater than g5%.
Example 7 Treatment of a pure 2% silyl-CSP support with 150 ug of Polybrene greatly reduced the retention of the peptlde and thus the yield of the PTH derivatives of the analyzed decapeptlde. But if 1.5 mg of Polybrene is added, the retention of peptide is comparable to that of Polybrene treated underivatized glass supports.
If the silyl-CSP support worked by embedding the sample, then adding a small amount of additional embedding agent (i.e., Polybrene) should not result in a loss of sample retention. If, however, the Porton support works on a principle based on electrostatic interaction with the sample, addlng a small amount of a polybase (i.e., positively charged) substance such as Polybrene would very likely lnterfere with the electrostatic attraction of the negatlvely charged support for the sample. This small amount of polybase ls not sufficient, however, to embed the sample. Adding more Polybrene (~1.5 mg) will completely cover the charged groups on the support and embed the sample.

Claims (23)

1. In a method of sequencing a peptide or protein which comprises immobilizing the peptide or protein on a support and sequencing the peptide or protein by stepwise degradation, the improvement which comprises providing a glass support derivatized with a silica-binding substance having a free acid group, said acid having a pKa<1, said derivatized support being capable of retaining said peptide or protein.

2. The method of claim 1, wherein said acid group is selected from the group consisting of phosphoric, picric, hydrochloric, trifluoroacetic, trichloroacetic, chromic, hydroiodic, pyrophosphoric, sulfonic and halosulfonic acids.

3. The method of claim 1 in which the acid group is a sulfonic or halosulfonic acid.

4. The method of claim 3 in which the support is prepared by incubating a glass support with a 4-chlorosulfonyl phenyl alkyl alkoxysilane.

5. The method of claim 4 in which the silane is 2-(4-chlorosulfonyl phenyl) ethyl trimethoxysilane.

6. The method of claim 4 in which the silane is 3-(trimethylsilyl)-l-propane sulfonic acid.

7. The method of claim 1, wherein the silica-binding substance is an organosilane having the formula RnSiX(4-n), where n is 1 to 3, X is a hydrolyzable group suitable for covalently binding the substance to the glass support, and R
is a nonhydrolyzable organic radical bearing said free acid group, said derivatized support being capable of retaining the peptide or protein.

8. The method of clalm 7, wherein X is selected from the group consisting of alkoxy, acyloxy, amine and chlorine.

9. In a method of sequencing a peptide or protein which comprises immobilizing the peptide or protein on a support and sequencing the peptide or protein by stepwise degradation, the improvement which comprises providing a glass support derivatized with a substance which covalently binds to silica, said substance having a quaternary ammonium group, said derivatized support being capable of retaining the peptide or protein.

10. The method of claim 9 in which the substance is an organosilane.

11. The method of claim 10 in which the substance is n-trimethoxysilyl propyl-N,N,N-trimethyl ammonium chloride.

12. The method of claim 10, wherein the organisilane has the formula RnSiX(4-n), where n is 1 to 3, X is a hydrolyzable group suitable for covalently bonding the substance to the glass support, and R is a nonhydrolyzable organic radical bearing a quaternary ammonium group, said derivatized support being capable of retaining the peptide or protein.

13. The method of claim 12, wherein X is selected from the group consisting of alkoxy, acyloxy, amine and chlorine.

14. A derivatized glass support comprising a silicaceous support derivatized with a first silica-binding substance having a free acid group, said acid group having a pKa<1, and a second silica-binding substance having a free quaternary ammonium group, said support being capable of retaining a peptide or protein.

15. The support of claim 14, wherein said acid group is selected from the group consisting of phosphoric, picric, hydrochloric, trifluoroacetic, trichloroacetic, chromic, hydroiodic, pyrophosphoric, sulfonic and halosulfonic acids.

16. The support of claim 14 in which the acid group is a sulfonic or halosulfonic acid.

17. The support of claim 16 in which the support is prepared by incubating a glass support with a 4-chlorosulfonyl phenyl alkyl alkoxysilane.

18. The support of claim 17 in which the silane is 2-(4-chlorosulfonyl phenyl) ethyl trimethoxysilane.

19. The support of claim 17 in which the silane is 3-(trimethylsilyl)-1-propane sulfonic acid.

20. The support of claim 14 in which the first substance is 2-(4-chlorosulfonyl phenyl) ethyl trimethoxysilane, and the second substance is N-trimethoxysilyl propyl-N,N,N-trimethyl ammonium chloride.

21. The support of claim 14 in which the first or second substance is an organosilane.

22. The support of claim 21 in which the second substance is N-trimethoxysilyl propyl-N,N,N-trimethyl ammonium chloride.

23. In a method of sequencing a peptide or protein which comprises immobilizing the peptide or protein on a support and sequencing the peptide or protein by stepwise degradation, the improvement which comprises immobilizing the peptide or protein on the support of any one of claims 14 to 22.