Journal of Chromatography, 530 (1990) 29-37
`Biomedical Applications
`Elsevier Science Publishers B.V., Amsterdam
`
`CHROMBIO. 5344
`
`High-performance liquid chromatography of rat and mouse
`islet polypeptides: potential risk of oxidation of methionine
`residues during sample preparationa
`
`S. LINDE*, J. H. NIELSEN, B. HANSEN and B. S. WELINDER
`
`Hagedorn Research Laboratory, Niels Steensensvej 6, DK-2820 Gentofte (Denmark)
`
`(First received November 3rd, 1989; revised manuscript received March 26th, 1990)
`
`ABSTRACT
`
`After preparative high-performance liquid chromatography of mouse islet culture medium, concentrat(cid:173)
`ed on disposable C 18 cartridges (Sep-Pak), an unexpected insulin immunoreactive peak eluting earlier than
`mouse insulin I and II was detected. Molecular mass determination by mass spectrometry supported its
`suspected identity as methionine sulphoxide insulin II. We have examined the formation of Met-0 deriv(cid:173)
`atives of insulin II, glucagon and pancreatic polypeptide during sample preparation (Sep-Pak and Speed(cid:173)
`Vac concentrating). The oxidation of methionine residues was found to depend very much on the buffer,
`the organic modifier and the procedure. In particular the use of methanol-trifluoroacetic acid resulted in
`extensive oxidation. The oxidation could be minimized by adding 2 mM dithiothreitol to the buffer and by
`degassing and/or nitrogen-bubbling of the buffer. Minimal formation of Met-0 derivatives is important for
`the quantitation of methionine-containing polypeptides.
`
`INTRODUCTION
`
`Studies of the biosynthesis of proinsulins and their conversion into insulins
`and C-peptides in the rat and mouse endocrine pancreas depend on accessible
`reversed-phase high-performance liquid chromatographic (RP-HPLC) analyses
`capable of separating all the polypeptides involved.
`We have recently described two HPLC systems that can separate the two
`non-allelic insulins (I and II) from rat and mouse, as well as the two C-peptides (I
`and II), and the two proinsulins (I and II) [l]. In order to identify the individual
`mouse polypeptides by amino acid analysis and micro-sequencing [1] we concen(cid:173)
`trated medium from cultured mouse islets on disposable C1 8 cartridges (Sep(cid:173)
`Pak). In some cases this procedure resulted in an unexpected peak which reacted
`as insulin by radioimmunassay after RP-HPLC separation of the concentrated
`medium. Since insulin IL in contrast to insulin I, contains a methionine residue in
`
`a Presented in part at the 9th International Symposium on HPLC of Proteins. Peptides and Polynucleo(cid:173)
`tides, Philadelphia, PA. November 6-8, 1989. The majority of the papers presented at this symposium have
`been published in J. Chromatogr., Vol. 512 (1990).
`
`03 78-434 7 /90/$03.50 © 1990 Elsevier Science Publishers B. V.
`
`CFAD Exhibit 1052
`CFAD v. NPS
`IPR2015-00990
`
`

`
`30
`
`S. LINDE et al.
`
`position 29 of the B-chain, oxidation to methionine sulphoxide in insulin II might
`have happened during sample preparation [2-5].
`The aim of the present study was to investigate the influence of buffer compo(cid:173)
`nents, organic modifiers and isolation procedures on the formation of Met-0
`derivatives of insulin II, as well as of glucagon and pancreatic polypeptide, in
`order to minimize the oxidation of the methionine residues.
`
`EXPERIMENT AL
`
`Reagents
`Trifluoroacetic acid (TF A, Peptide Synthesis grade) was from Applied Bio(cid:173)
`systems and acetonitrile (HPLC grade S) was from Rathburn. All other chemicals
`were of analytical-reagent grade. Distilled water was purified on a Millipore Mil(cid:173)
`li-Q plant, and all buffers were filtered (0.45 µm, Millipore) and degassed by
`vacuum/ultrasound before use.
`
`Standards
`As a source of rat insulin I and II and C-peptide I and II, medium from
`newborn rat islet cells cultured in RPMI 1640 supplemented with 2% human
`serum in the presence of I µg/ml human growth hormone was used. The medium
`contained 44 µg/ml of insulin I and II and equimolar amounts of C-peptide I and
`II. Rat pancreatic polypeptide was obtained from Penninsula and porcine gluca(cid:173)
`gon from Sigma. Rat insulin II was prepared from the culture medium using
`preparative HPLC.
`
`Chromatography
`The HPLC system consisted of Waters M6000 A pumps, a WISP 710A, a 660
`solvent programmer, a 730 data module and a Pye Unicam LC-UV detector. The
`column was LiChrosorb RP-18, 5 µm particle size, 250 x 4.0 mm l.D. (Merck),
`eluted at 1.0 ml/min with a linear gradient of acetonitrile (30 to 36%) in O. l %
`TFA during 60 min. The column eluate was monitored at 210 nm, peaks were
`collected either manually or by collecting 0.5-min fractions in a FRAC 300 frac(cid:173)
`tion collector (Pharmacia). All separations were carried out at room temperature.
`
`Sample preparation using Sep- Pak
`Disposable C 1s cartridges (Sep-Pak, Waters) were used. The organic eluent
`(B) contained 90% (v/v) of the organic modifier, which was acetonitrile, 2-propa(cid:173)
`nol or methanol. The aqueous eluent (A) was water, 1 M acetic acid or 0.1 %
`TFA. The Sep-Pak cartridge was flushed with IO ml of Band 10 ml of A, and the
`sample was applied, followed by washing with IO ml of A and elution with 1.5 ml
`of B into an Eppendorf tube. The sample was concentrated in a Speed-Vac con(cid:173)
`centrator (Savant).
`
`

`
`HPLC OF RAT AND MOUSE ISLET POLYPEPTIDES
`
`31
`
`Radioimmunoassay (RIA)
`Collected fractions containing TFA-acetonitrile were dried in a Speed-Vac
`concentrator, and radioimmunological determination of insulin was carried out
`using rat insulin (Novo) as a standard and anti-mouse insulin antibodies (devel(cid:173)
`oped in this laboratory) as previously described [6].
`
`Hydrogen peroxide oxidation
`A 1-ml volume of a solution containing 1 % hydrogen peroxide (v/v) in 3 M
`acetic acid was evaporated in the Speed-Vac concentrator in a separate tube,
`together with 2-40 µg samples of glucagon, pancreatic polypeptide or insulin II
`dissolved in 3M acetic acid, as well as rat culture medium diluted 1:1 (v/v) with 3
`M acetic acid.
`
`Determination of molecular mass
`The molecular masses (Mr) of the polypeptides and their oxidized forms isolat(cid:173)
`ed after HPLC were determined by mass spectrometry (MS). The mass spectra
`were obtained on a Bio-Ion 20 plasma desorption mass spectrometer [7]. Prior to
`analysis the samples were applied to nitrocellulose targets prepared on Mylar foil.
`Spectra were accumulated for 5· l 06 fission events.
`
`RES UL TS AND DISCUSSION
`
`Isolation and identification of polypeptides and proteins present in very small
`amounts in complex solutions, such as cell culture media, normally require a
`concentration step before the HPLC analysis, and especially for large volumes it
`is convenient to use disposable Sep-Pak C1s cartridges for sample preparation.
`Mouse islet culture media (100 ml) were loaded on a Sep-Pak, the cartridge
`was washed with 10 ml of 30% methanol (thereby eluting several non-peptide
`culture medium constituents) and thereafter the polypeptides were eluted with
`90% methanol. Fig. 1 shows the HPLC analysis of the concentrated medium.
`Although several sample components with no relevance to the C-peptides and
`insulins were removed before HPLC, the presence of numerous UV-absorbing
`components made a direct localization of the islet polypeptides difficult: mouse
`C-peptide I and II, expected to elute at 25-30 min, could not be distinguished
`from neighbouring components, and only after insulin RIA were the insulins
`localized in three peaks. Peaks 2 and 3 eluted at the same retention times as rat
`insulin I and II, respectively (rat and mouse insulins are identical).
`In order to verify the hypothesis that insulin peak 1 could be methionine
`sulphoxide insulin II, rat islet culture medium was oxidized with hydrogen perox(cid:173)
`ide reported to oxidize methionine to methionine sulphoxide [8] as described
`above. HPLC analysis of this sample showed that an additional component with
`a lower retention time (marked with an arrow, Fig. 2) was formed during ox(cid:173)
`idation simultaneously with a decrease of the amount of insulin II. The lower
`
`

`
`32
`
`0.08
`
`0
`
`S. LINDE et al.
`
`600
`
`c
`.2
`400 u
`~
`"' .5.
`.!:
`3
`"' £
`
`200
`
`0
`80
`
`40
`
`Elution time (min)
`
`Fig. I. HPLC separation of 40 ml of Sep-Pak-concentrated mouse islet culture medium using a LiChrosorb
`RP-18 column (250 x 4.0 mm l.D.) eluted at a flow-rate of 1.0 ml/min with a concave acetonitrile gradient
`(gradient 7, 30 to 36%) in 0.1% TFA during 60 min; 0.5-min fractions were collected and dried in a
`Speed-Vac, and the insulin content was determined using RIA (histogram).
`
`0.08
`
`~
`uS
`
`0
`
`0.08
`
`Rat islet culture medium
`
`c
`]
`c
`
`c
`
`w
`"
`w ~
`"' Q
`c.
`' w (.)
`?-
`
`(.)
`
`Oxidized culture medium
`
`0
`
`0
`
`20
`
`40
`
`60
`
`Elution time (min)
`Fig. 2. HPLC separation of 50 µI of rat islet cell culture medium and 75 µI of H 2 0 2-oxidized medium using
`a LiChrosorb RP-18 column (250 x 4.0 mm I.D.) eluted at a flow-rate of 1.0 ml/min with a linear aceto(cid:173)
`nitrile gradient (30-36%) in 0.1 % TFA during 60 min.
`
`

`
`HPLC OF RAT AND MOUSE ISLET POLYPEPTIDES
`
`33
`
`retention time was expected since the Met-0 derivatives are reported to have
`higher polarity [IO]. The retention time of this newly formed component relative
`to that of insulin II could not directly be compared with that of peak 1 in Fig. 1,
`since different acetonitrile gradients were used (see legends to Figs. 1 and 2). The
`identification of rat insulin I and II (Fig. 2, upper panel) was based on amino acid
`analysis and sequencing, as well as by RIA as previously described [l].
`Likewise, oxidation of other methionine-containing islet polypeptides resulted
`in additional peaks with lower retention times as shown for glucagon (Fig. 3, two
`additional peaks) and pancreatic polypeptide (Fig. 4). The additional peaks
`(marked with arrows in Figs. 2-4) as well as the authentic polypeptides were
`collected after HPLC and subjected to MS. If necessary, the samples were con(cid:173)
`centrated in the Speed-Vac concentrator before MS. The resulting molecular
`masses are shown in Table I, together with the theoretically calculated values. In
`the case of glucagon, two additional peaks were detected (Fig. 3) that had the
`same molecular mass (Table I), although only one methionine residue is present.
`Two different methionine sulphoxide forms exist. the D- and L-forms. but it is
`
`0.08
`
`0
`
`0.08
`
`0
`
`Glucagon
`
`Oxidized glucagon
`
`1 2
`
`H
`
`20
`
`4o
`
`a'o
`
`Elution time (min)
`
`Fig. 3. HPLC separation of glucagon (2 µg) and H 20 2-oxidized glucagon (3 pg). Conditions as in Fig. 2.
`
`

`
`34
`
`0.08
`
`C> w
`
`0
`
`0.08
`
`~
`N w
`
`0
`
`0
`
`Rat pancreatic polypeptide (PP)
`
`S. LINDE et al.
`
`Oxidized rat PP
`
`20
`
`40
`
`60
`
`Elution time (min)
`
`Fig. 4. HPLC separation of rat pancreatic polypeptide (1 µg) and H 20 2-oxidized pancreatic polypeptide (2
`µg). Conditions as in Fig. 2.
`
`TABLE I
`
`RELATIVE MOLECULAR MASSES OF POLYPEPTIDES AND OXIDIZED POLYPEPTIDES DE-
`TERMTNED BY MS
`
`M, found
`
`M, calculated"
`
`Insulin II
`Oxidized insulin TI
`
`Glucagon
`Oxidized glucagon (1)
`Oxidized glucagon (2)
`
`Pancreatic polypeptide (PP)
`Oxidized PP
`
`5801.6
`5818.5
`
`3485.7
`3503.1
`3501.9
`
`4396.4
`4416.9
`
`5796
`5812
`
`3483
`3499
`3499
`
`4399
`4415
`
`"The calculated molecular masses of the Met-0 derivatives were derived by adding 16 to the known
`molecular masses of the individual polypeptides.
`
`

`
`HPLC OF RAT AND MOUSE ISLET POLYPEPTIDES
`
`35
`
`TABLE II
`
`AMOUNTS OF OXIDIZED GLUCAGON (G), PANCREATIC POLYPEPTIDE (PP) AND INSULIN
`II (I 2 ) DERIVATIVES FORMED DURING SAMPLE PREPARATION
`
`Solvents: AcOH = acetic acid; MeOH = methanol; isoPrOH = 2-propanol: MeCN = acetonitrilc.
`
`Sample
`preparation
`
`Solvent
`
`Oxidized G Oxidized PP Oxidized I 2
`(%)a
`(%)"
`(%)"
`
`Speed-Vac
`Speed-Vac
`Speed-Vac
`Speed-Vac
`Speed-Vac
`Speed-Vac
`Speed-Vac
`Speed-Vac
`Speed-Vac
`Sep-Pakb
`Sep-Pak
`Sep-Pak
`Sep-Pak
`Sep-Pak
`Sep-Pak
`Sep-Pak
`Sep-Pak
`Sep-Pak
`
`Water
`3M AcOH
`0.1% TFA
`90% MeOH-1 M AcOH
`90% MeOH-0.1% TFA
`90% MeCN-1 M AcOH
`90% MeCN-0. l % TF A
`90% isoPrOH-1 M AcOH
`90% isoPrOH--0.1 % TF A
`90% MeOH-H 20
`90% MeOH-1 M AcOH
`90% MeOH--0.1 % TF A
`90% MeCN-H 2 0
`90% MeCN-lM AcOH
`90% MeCN--0.1 % TF A
`90% isoPrOH-H 20
`90% isoPrOH-1 M AcOH
`90% isoPrOH--0. I% TFA
`
`0
`0
`6
`33
`2
`2
`
`6
`5
`63
`0
`9
`3
`17
`24
`42
`
`"Calculated from UV areas (210 nm) after HPLC.
`b As described in Experimental.
`
`0
`0
`8
`42
`3
`4
`6
`8
`16
`17
`89
`0
`14
`25
`16
`18
`27
`
`0
`2
`4
`
`51
`3
`2
`14
`12
`44
`46
`79
`31
`51
`47
`29
`40
`63
`
`unlikely that these two forms could be separated in the present HPLC system.
`Since the Mr values of these peaks were higher (ca. 16) than that of the parent
`compounds and since the formation of Met-0 derivatives of rat and mouse in(cid:173)
`sulin II and proinsulin II during storage and sample treatment [2---4], as well as of
`other methionine-containing polypeptides [11-14], has been described to occur
`under similar conditions to those reported here, they most probably represent
`Met-0 derivatives of insulin II, glucagon and PP. In the case of glucagon and
`corticotropin-releasing factor, the formation of Met-0 derivatives results in re(cid:173)
`duced biological activity [11, 12].
`The formation of Met-0 derivatives was found to depend very much on the
`actual procedure and buffers, as well as the Sep-Pak cartridge used (Table II). In
`particular the use of 0.1 % TF A-methanol resulted in extensive modification.
`Furthermore, the amount of modified polypeptide was inversely related to the
`amount of polypeptide used (exemplified by glucagon): Sep-Pak purification us(cid:173)
`ing TFA-acetonitrile of 4, 10 and 40 µg resulted in 23, 7 and 3% of oxidized
`glucagon derivatives, respectively. In accordance with published results [4, 15] for
`pre-purification of transfected AtT20 cells. we observed that the use of TFA-
`
`

`
`36
`
`TABLE Ill
`
`S. LINDE et al.
`
`AMOUNTS OF OXIDIZED GLUCAGON (G), PANCREATIC POLYPEPTIDE (PP) AND INSULIN
`IT (T 2 ) DERIVATIVES FORMED DURING SAMPLE PREPARATION WITH ADDITION OF 2 mM
`ASCORBIC ACID (AA) OR 2 mM DITHIOTREITOL (DTT)
`
`Sample
`preparation
`
`-~-~~
`
`Speed-Vac
`Speed-Vac
`Speed-Vac
`Speed-Vac
`Speed-Vac
`Speed-Vac
`Sep-Pakb
`Sep-Pak
`Sep-Pak
`
`Solvent
`
`Additive
`
`Oxidized
`(%)"
`
`Oxidized PP
`(%)"
`
`Oxidized 12
`(%)"
`
`3M AcOH
`3M AcOH
`3M AcOH
`90% MeOH--0.1 % TF A
`90% MeOH--0.1% TFA
`90% MeOH-0.1% TFA
`90% MeOH--0.1 % TF A
`90% MeOH--0.1 % TF A
`90% MeOH--0.1% TFA
`
`AA
`DTT
`
`AA
`DTT
`
`AA
`DTT
`
`0
`
`33
`40
`0
`63
`41
`7
`
`0
`
`42
`47
`3
`61
`21
`13
`
`2
`
`51
`
`28
`79
`
`8
`
`"Calculated from UV areas (210 nm) after HPLC.
`b As described in Experimental.
`
`acetonitrile-methylene chloride, resulted in extensive formation of Met-0 deriv(cid:173)
`atives (data not shown).
`In order to minimize the oxidation, we incorporated 2 mM ascorbic acid (AA)
`or 2 mM dithiothreitol (DTT) in the buffers during sample preparation (Table
`III). Only DTT effectively protects the methionine group, in accordance with
`published results [16). The Met-0 formation could also be decreased by degassing
`the buffers. This effect was further enhanced by additional bubbling with nitrogen
`(data not shown).
`In conclusion, we have shown that Speed-Vac drying and Sep-Pak sample
`preparation may result in formation of methionine sulphoxide derivatives. The
`amount of transformation product depends very much on the actual Sep-Pak
`used and the time for the sample preparation. Thus the amounts reported here
`can only serve as an indication. In order to minimize the oxidative transformation
`of methionine we recommend the addition of 2 mM DTT to prevent the HPLC
`chromatograms being misleading, and to avoid the reduction of biological activ(cid:173)
`ity.
`
`ACKNOWLEDGEMENTS
`
`We thank Dr. Stephen Bayne (Novo-Nordisk A/S) for performing the molec(cid:173)
`ular mass determinations, and Ragna J0rgensen for excellent technical assistance.
`
`

`
`HPLC OF RAT AND MOUSE ISLET POLYPEPTIDES
`
`37
`
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`
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