Supplementary Information For s4

Supplementary information for:

‘Probing the transient interaction between the small heat shock protein Hsp21 and a model substrate protein using crosslinking mass spectrometry’, by Wietske Lambert, Gudrun Rutsdottir, Rasha Hussein, Katja Bernfur, Sven Kjellström, and Cecilia Emanuelsson.

Department of Biochemistry and Structural Biology, Center for Molecular Protein Science, Institute for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden

Contents:

Figure S-1

Table S-1

Table S-2

Table S-3

Table S-4

Figure S-2

Figure S-3

Figure S-1. Substrate protein heat-treated in absence and presence of Hsp21 analyzed by native-PAGE. Hsp21 and substrate protein CS (citrate synthase) were pre-incubated alone or mixed in 1/1 molar ratio and directly loaded on native-PAGE (Native-PAGE Phastgels, 8-25% gradient, Tris-Alanine buffer, pH 8.8, GE Healthcare) and the native-PAGE run at 25°C, or at 45°C where indicated. MW, molecular weight marker; Lanes 1-3, Hsp21 only, CS only, and Hsp21 and CS, respectively, pre-incubated 30 min at 25°C; Lanes 4-6, as Lanes 1-3, pre-incubated 30 min at 45°C; Lanes 7-9, as Lanes 1-3, pre-incubated 30 min at 45°C and gel run at 45°C. The large stable complexes formed between Hsp21 and substrate protein CS pre-incubated for 30 min at 45°C and run at 45°C cannot enter into the gel and remain on the loading spot (Lane 9). No stable complexes are formed by pre-incubation for 30 min at 45°C, since Hsp21 dodecamers and CS dimers run separately (Lane 6), just as in non-heated sample (Lane 3). Pre-incubation of CS 30 min at 45°C in absence of Hsp21 leads to aggregation and no detection on gel.

Comment on size fractionation in native-PAGE. Below is a general comment regarding size fractionation in native-PAGE, and a specific comment regarding why the better performing native-PAGE system used here in figure S-1 could be used for analysis of the substrate protein CS (pI = 7.0), but not for MDH (pI = 8.6), for which the less well performing native-PAGE system shown in figure 2 was used.

In SDS-PAGE proteins are denatured by SDS, which is breaking non-covalent bonds and dissociating oligomers by binding to protein (on average 1 SDS per two amino acids), resulting in a high negative charge to all proteins, irrespective of their intrinsic charge. Proteins therefore rapidly migrate as monomeric sharp bands towards the positively charged anode, with the protein mobility being inversely proportional to the monomer size. Contrastingly, in native PAGE, non-denatured oligomeric proteins migrate based on their intrinsic charge, which depends on the protein pI, and the pH during the separation. Highly charged proteins have higher mobility than less charged proteins, which migrate slower. Adequate size fractionation in native PAGE can be obtained if two criteria are fulfilled, a broad gradient of polyacrylamide (such that the pore size gradually gets smaller and smaller along the gel in which proteins migrate up to the point where the pore size is smaller than the protein size) and a long enough running time (such that also slowly migrating lowly charged proteins reach to and get caught at that point). In our experience (e.g. Härndahl et al 1998, Protein Expr Purif. 14, 87-96) manually cast gels with 3-27% gradient and a discontinuous Laemmli Tris-Glycine buffer fulfill these criteria when run over-night (proteins do not run out into buffer by a long run-time, as in SDS-PAGE, since they are caught at their respective mass positions due to the narrowing pore size). Easier to use, yet with a tradeoff in terms of lower performance, are commercially available ready-made gels with short run-times. We routinely use two such systems, Native-PAGE Phastgel™ (8-25% gradient, Tris-Alanine buffer, pH 8.8, GE Healthcare, Uppsala, Sweden) and NativePAGE™ Novex® Bis-Tris gels (3-12% gradient, Bis-Tris buffer pH 7.6, Life Technologies Europe BV, Stockholm, Sweden).

The Native-PAGE Phastgel™ system (see figure S-1) gives very sharp bands to most proteins, with pI < 8.8. For example Hsp21 (pI ≈ 5.7) is negatively charged at pH 8.8. The substrate protein CS (CS, pI≈7.0) readily migrates towards the anode since it has an intrinsic charge that is negative at pH 8.8. It can be noted that compared to the Hsp21 oligomer (pI ≈5.7) the CS dimer (pI ≈7.0) is less negatively charged at pH 8.8, presumably explaining why CS is appearing as a band higher up than Hsp21, since after the standard run-time used (250 so called AVh, Accumulated Volt hours) it has not yet reached the position where the pore size of the polyacrylamide gradient prevents it from proceeding further. However, unfortunately the unusually high pI-value of MDH (pI≈8.6) means that it is uncharged or even slightly positively charged at pH 8.8 and MDH does not enter into the gel in this system.

The NativePAGE™ Novex® Bis-Tris gels system (see figure 2) was instead used for MDH. According to the manufacturer’s instructions, the running buffer is supplied with Coomassie Brilliant Blue stain G-250 which binds to proteins contributing to a net negative charge without denaturation. Yet, in case of MDH, for which the intrinsic charge at pH 7.6 is positive, the G-250 negative charging is not enough to make it focus into sharp bands. Still, even in this low-performance gel system it is possible to evaluate when MDH is complexed to Hsp21 or not.

Table S-1. Hsp21-Hsp21 crosslinks identified. Criteria for confident identification: search settings of data analysis program FINDX as defined in materials and methods, and unambiguous assignment of the theoretical mass to the proposed peptide pair (within 60 ppm).

Theoreti-cal (MH)+ (Da) / Matched Hsp21
sequence 1 / Matched Hsp21
sequence 2 / Hsp21 res. 1 / Hsp21 res. 2 / Obser-vations / Ox. Met obser-vationa
1491.6684 / M1QDQR5 / M1QDQR5 / 1 / 1 / 4 / 2
2385.1693 / M1QDQR5 / G19NQGSSVEKRPQQR32 / 1 / 27 / 7 / 1
2438.1662 / M1QDQR5 / A84PWDIKEEEHEIK96 / 1 / 89 / 4
2180.0367 / M1QDQR5 / F99DMPGLSKEDVK110 / 1 / 106 / 1
2095.8991 / M1QDQR5 / K126EDSDDSWSGR136 / 1 / 126 / 1
1758.9424 / M1QDQR5 / D154KIKAELK161 / 1 / 155 / 3 / 1
1515.8205 / M1QDQR5 / I156KAELK161 / 1 / 157 / 5 / 1
1446.7375 / M1QDQR5 / T172KVER176 / 1 / 173 / 4 / 1
1574.8324 / M1QDQR5 / T172KVERK177 / 1 / 173 / 1
1756.9267 / M1QDQR5 / K177VIDVQIQ184 / 1 / 177 / 2 / 1
3128.5824 / E6NSIDVVQQGQQKGNQGSSVEK27 / T172KVER176 / 18 / 173 / 7
2297.1962 / G19NQGSSVEKRPQQR32 / G122EQKK126 / 27 / 125 / 3
2211.1846 / G19NQGSSVEKRPQQR32 / D154KIK157 / 27 / 155 / 1
2340.2384 / G19NQGSSVEKRPQQR32 / T172KVER176 / 27 / 173 / 7
2393.2352 / A84PWDIKEEEHEIK96 / T172KVER176 / 89 / 173 / 1
1970.0017 / E90EEHEIKMR98 / T172KVER176 / 96 / 173 / 8
2098.0966 / E90EEHEIKMR98 / T172KVERK177 / 96 / 173 / 1
2833.5425 / F99DMPGLSKEDVK110 / N162GVLFITIPKTK173 / 106 / 171 / 4 / 1
2440.3662 / I111SVEDNVLVIKGEQK125 / T172KVER176 / 121 / 173 / 1
2379.2052 / G122EQKK126 / M97RFDMPGLSKEDVK110 / 125 / 106 / 5
1668.9536 / G122EQKK126 / K177VIDVQIQ184 / 125 / 177 / 5
1272.7527 / D154KIK157 / T172KVER176 / 155 / 173 / 1
1400.8477 / D154KIK157 / T172KVERK177 / 155 / 173 / 1
1470.8895 / I156KAELK161 / T172KVER176 / 157 / 173 / 2

aThe observation(s) of the same peptide pair but with one or two methionine residues oxidized, which strengthens the identification. This type of observation was only counted once, even if it was observed in several data sets.

Table S-2. MDH-MDH crosslinks identified. Criteria for confident identification: search settings of data analysis program FINDX as defined in materials and methods, and unambiguous assignment of the theoretical mass to the proposed peptide pair (within 60 ppm).

Theoreti-cal (MH)+ (Da) / Matched MDH
sequence 1 / Matched MDH
sequence 2 / MDH res. 1 / MDH res. 2 / Obser-vations / Ox. Met obser-vationa
1411.7698 / A1K2 / K133HGVYNPNK141 / 1 / 133 / 2
1927.0653 / A1K2 / A153NAFVAELKGLDPAR167 / 1 / 161 / 4
1627.9270 / A1K2 / I206QEAGTEVVKAK217 / 1 / 215 / 5
1436.7459 / A1K2 / G306EEFVKNMK314 / 1 / 311 / 3 / 1
1882.9962 / K81PGMTR86 / K133HGVYNPNK141 / 81 / 133 / 7
2099.1534 / K81PGMTR86 / I206QEAGTEVVKAK217 / 81 / 215 / 11 / 1
2480.2754 / K81PGMTR86 / A216KAGAGSATLSMAYAGAR233 / 81 / 217 / 3
1662.8889 / K81PGMTR86 / K305GEEFVK311 / 81 / 305 / 7 / 1
2275.1545 / K133HGVYNPNK141 / G306EEFVKNMK314 / 133 / 311 / 5 / 1
2854.5831 / H134GVYNPNKIFGVTTLDIVR152 / K273GIEK277 / 141 / 273 / 7
3026.5984 / A216KAGAGSATLSMAYAGAR233 / A301SIKKGEEFVK311 / 217 / 304 / 6
1809.9638 / K305GEEFVK311 / K305GEEFVK311 / 305 / 305 / 4
2183.1422 / K305GEEFVK311 / K305GEEFVKNMK314 / 305 / 305 / 2

Table S-3. Type 0 and type 1 Hsp21 crosslinks identified. Criteria for confident identification: search settings of data analysis program FINDX as defined in materials and methods, and unambiguous assignment of the theoretical mass to the proposed peptide pair (within 60 ppm).

Theoreti-cal (MH)+ (Da) / Matched Hsp21
sequence / Crosslink type / Hsp21 residue / Obser-vations / Ox. Met obser-vationa
2515.2276 / E6NSIDVVQQGQQKGNQGSSVEK27 / 0 / 18 / 8
3173.5133 / M1QDQRENSIDVVQQGQQKGNQGSSVEK27 / 0 / 18 / 13 / 1
1726.8836 / G19NQGSSVEKRPQQR32 / 0 / 27 / 16
1779.8805 / A84PWDIKEEEHEIK96 / 0 / 89 / 5
2049.0115 / A84PWDIKEEEHEIKMR98 / 1 / 89, 96 / 3
2067.0221 / A84PWDIKEEEHEIKMR98 / 0 / 89/96 / 9
1356.6469 / E90EEHEIKMR98 / 0 / 96 / 2
1808.8926 / M97RFDMPGLSKEDVK110 / 0 / 106 / 7
1437.6134 / K126EDSDDSWSGR136 / 0 / 126 / 4
2280.2338 / E107DVKISVEDNVLVIKGEQK125 / 1 / 110, 121 / 4

Table S-4. Type 0 and type 1 MDH crosslinks identified. Criteria for confident identification: search settings of data analysis program FINDX as defined in materials and methods, and unambiguous assignment of the theoretical mass to the proposed peptide pair (within 60 ppm).

Theoreti-cal (MH)+ (Da) / Matched MDH
sequence / Crosslink type / MDH residue / Obser-vations / Ox. Met obser-vationa
2148.3007 / A1KVAVLGASGGIGQPLSLLLK21 / 0 / 2 / 2
2901.7141 / A1KVAVLGASGGIGQPLSLLLKNSPLVSR28 / 0 / 2/21 / 6
2702.5820 / V3AVLGASGGIGQPLSLLLKNSPLVSR28 / 0 / 21 / 9
845.4555 / K81PGMTR86 / 0 / 81 / 14 / 1
2108.1360 / G68CDVVVIPAGVPRKPGMTR86 / 0 / 81 / 9 / 1
1212.6377 / K133HGVYNPNK141 / 0 / 133 / 9
2299.2450 / H134GVYNPNKIFGVTTLDIVR152 / 0 / 141 / 15
1727.9332 / A153NAFVAELKGLDPAR167 / 0 / 161 / 15
1428.7950 / I206QEAGTEVVKAK217 / 0 / 215 / 10
2864.4828 / I206QEAGTEVVKAKAGAGSATLSMAYAGAR233 / 0 / 215/217 / 2
2748.5057 / S256QETDCPYFSTPLLLGKKGIEK277 + 1 linker / 1 / 272, 273 / 1
1294.7734 / K273GIEKNLGIGK283 / 1 / 273, 277 / 8
1184.6890 / G274IEKNLGIGK283 / 0 / 277 / 6
1587.8634 / N278LGIGKISPFEEK290 / 0 / 283 / 5
2101.1142 / I284SPFEEKMIAEAIPELK300 / 0 / 290 / 5 / 1
1669.9450 / M291IAEAIPELKASIK304 / 0 / 300 / 2 / 1
1780.0294 / M291IAEAIPELKASIKK305 / 1 / 300, 304 / 4 / 1
1798.0399 / M291IAEAIPELKASIKK305 / 0 / 300/304 / 2
1373.7680 / A301SIKKGEEFVK311 / 1 / 304, 305 / 11
1764.9569 / A301SIKKGEEFVKNMK314 / 0 / 304/305/311 / 4 / 1
992.5304 / K305GEEFVK311 / 0 / 305 / 4

Figure S-2. (A-P) MS spectra of the Hsp21-MDH crosslinks listed in table 1. The peak labels shown are the masses based on default calibration, explaining the mass deviation of > 20 ppm in some cases.

1503.7412 0, 2, 1 M1QDQR5, K81PGMTR86 Hsp21, MDH

From file: 110722_Hsp21xMDH_lc4_ce-Well B19_35129.t2d

1519.7406 1, 2, 1 M1QDQR5, K81PGMTR86(1metox) Hsp21, MDH

From file: 110722_Hsp21xMDH_2_ce-Well B20_35127.t2d

1535.7400 0, 2, 1 M1QDQR5(1metox), K81PGMTR86(1metox) Hsp21, MDH

From file: 110722_Hsp21xMDH_lc4_ce-Well B19_35129.t2d

1870.9234 0, 2, 1 M1QDQR5, K133HGVYNPNK141 Hsp21, MDH

From file: 110722_Hsp21xMHD_2_ce-Well B20_35127.t2d

1886.9228 0, 2, 1 M1QDQR5(1metox), K133HGVYNPNK141 Hsp21, MDH

From file: 110722_Hsp21xMHD_2_ce-Well B20_35127.t2d

2087.0807 0, 2, 1 M1QDQR5, I206QEAGTEVVKAK217 Hsp21, MDH

From file: 110722_Hsp21xMHD_1_ce-Well F24_35126.t2d

2103.0801 0, 2, 1 M1QDQR5(1metox), I206QEAGTEVVKAK217 Hsp21, MDH

From file: Hsp21xMHD_lc_1-Well B9_35598.t2d

1388.7208 0, 2, 1 M1QDQR5, K273GIEK277 Hsp21, MDH

From file: 110722_Hsp21xMDH_lc4_ce-Well B13_35129.t2d

1404.7202 0, 2, 1 M1QDQR5(1metox), K273GIEK277 Hsp21, MDH

From file: 110722_Hsp21xMDH_lc4_ce-Well B13_35129.t2d

1842.9747 0, 2, 1 M1QDQR5, G274IEKNLGIGK283 Hsp21, MDH

From file: 110512_HM_B1-4-Well G12_34464.t2d

1360.7258 0, 2, 1 M1QDQR5, A301SIKK305 Hsp21, MDH

From file: 110622Hsp21xMDH-Well F19_34968.t2d