Table 1.
MALDI‐MS and μ‐HPLC/MALDI‐MS detection of −17 Da products of peptides having Gln and carboxyamidomethyl‐Cys residues at the N‐termini. All proteins were reduced, alkylated (iodoacetamide), and digested overnight with trypsin in solution, unless otherwise noted. MALDI‐MS spectra were collected for individual digests of 1 mg/mL proteins (0.3 mg/mL for integrin α5β1), or for portion of peptides extract after in‐gel digestion of SARS virus nucleocapsid protein. The data from two μ‐HPLC/MALDI‐MS runs have been analyzed: separation of 17‐protein digest mixture and separation of tryptic peptides from SARS virus nucleocapsid protein (see Experimental). Degree of conversion (%) has been calculated based on the ratio of monoisotopic peaks area (area product/(area product + area original peptide). M*—oxidized Met; D and D —Asn residues converted into Asp due to deamidation and deglycosylation with PNGase F, respectively; n/a—degradation product was not detected due to peak overlapping; δm —measured difference between unmodified and modified peptides
| Protein, peptide (start‐end) | MALDI‐MS | μ‐HPLC‐MALDI‐MS | ||||
|---|---|---|---|---|---|---|
| m/z measured | δm (Da) | Degree of conv. (%) | m/z measured | δm (Da) | Fraction # | |
| (A) N‐terminal Gln | ||||||
| SARS virus nucleocapsid protein, | 1183.585 | −17.027 | 28 | 1183.589 | −17.032 | 19 |
| QYNVTQAFGR (267–276) in‐gel | 1166.558 | 1166.557 | 21 | |||
| SARS virus nucleocapsid protein, | 1892.892 | −17.012 | 45 | 1892.905 | −17.026 | 23 |
| QPTVTLLPAADM*DDFSR (389–405) in‐gel | 1875.880 | 1875.879 | 24 | |||
| Human Integrin α5, | 1628.901 | n/a | n/a | 1628.902 | −17.032 | 22 |
| QATLTQTLLIQDGAR (567–581) | n/a | 1611.870 | 23 | |||
| Human Integrin α5, | 1131.614 | −17.026 | 31 | 1131.612 | −17.023 | 18 |
| QVATAVQWTK (981–990) | 1114.588 | 1114.589 | 20 | |||
| Phosphorylase B, | 1580.832 | −17.022 | 38 | 1580.832 | −17.023 | 26, 27 |
| QIIEQLSSGFFSPK (728–741) | 1563.810 | 1563.809 | 29 | |||
| Phosphorylase B, | 1756.956 | −17.017 | 26 | 1756.954 | −17.023 | 26, 27 |
| QLLNCLHVITLYNR (564–577) | 1739.939 | 1739.931 | 29 | |||
| Pepsinogen, | 1091.519 | −17.025 | 34 | 1091.520 | −17.024 | 20 |
| QYYTVFDR (352–359) | 1074.494 | 1074.496 | 22 | |||
| Bovine serum albumin, | 658.317 | −17.023 | 38 | 658.319 | −17.027 | 4 |
| QEPER (118–122) | 641.294 | 641.292 | 4 | |||
| (B) N‐terminal carboxyamidomethyl‐Cys | ||||||
| Human Integrin α5, | 1068.435 | −17.024 | 35 | 1068.429 | −17.029 | 15 |
| CPEAECFR (911–918) | 1051.409 | 1051.400 | 18 | |||
| Human Integrin α5, | 2307.146 | −17.019 | 45 | 2307.135 | −17.025 | 24 |
| CELGPLHQQESQSLQLHFR (921–939) | 2290.127 | 2290.110 | 25 | |||
| Human Integrin β1, CNEGR (491–495) | — | — | — | 635.260 | −17.025 | 4 |
| 618.235 | 4 | |||||
| Human Integrin β1, | 2242.053 | −17.025 | 51 | 2242.055 | −17.027 | 31 |
| CS D ISIGDEVQFEISITSNK (415–434) | 2225.028 | 2225.028 | 32 | |||
| Human Integrin β1, | 1655.599 | −17.020 | 60 | 1655.601 | −17.025 | 15 |
| CHEG D GTFECGACR (477–490) | 1638.579 | 1638.576 | 16 | |||
| Human serotransferrin, CQSFR (38–42) | — | — | — | 697.314 | −17.026 | 11 |
| 680.288 | 14 | |||||
| Human apo‐transferrin, | — | — | — | 1280.558 | −17.023 | 18 |
| CDEWSVNSVGK (374–384) | 1263.535 | 20 | ||||
| Human apo‐transferrin, | 1531.689 | −17.027 | 43 | 1531.694 | −17.025 | 22 |
| CSTSSLLEACTFR (684–696) | 1514.662 | 1514.669 | 23 | |||
| Bovine α‐lactalbumin, | 710.334 | −17.023 | 58 | 710.332 | −17.023 | 15 |
| CEVFR (6–10) | 693.311 | 693.309 | 17 | |||
| Chymotrypsinogen A bovine, | 1553.842 | −17.023 | 46 | 1553.850 | −17.029 | 25 |
| CGVPAIQPVLSGLSR (1–15) | 1536.819 | 1536.821 | 27 | |||
| Bovine serum albumin, | — | — | — | 706.358 | −17.023 | 10 |
| CASIQK (223–228), or human serum albumin | 689.335 | 12 | ||||
| CASLQK (197–202) | ||||||
| Bovine serum albumin, | 1138. 495 | −17.018 | 63 | 1138.503 | −17.025 | 15 |
| CCTESLVNR (499–507) or human serum albumin (473–481) | 1121.477 | 1121.478 | 16, 17 | |||
| Bovine serum albumin, | — | — | — | 1166.495 | −17.028 | 10, 11 |
| CCTKPESER (460–468) | 1149.467 | 12 | ||||
| Bovine serum albumin, | 1927.812 | n/a | n/a | 1927.798 | −17.028 | 18 |
| CCAADDKEACFAVEGPK (581–597) | n/a | 1910.770 | 19 | |||
| Bovine apo‐transferrin, CASFR (38–42) | — | — | — | 640.290 | −17.023 | 12 |
| 623.267 | 15 | |||||
| Bovine apo‐transferrin, | — | — | — | 1362.715 | −17.030 | 23 |
| CGLVPVLAENYK (424–435) | 1345.685 | 25 | ||||
| Bovine apo‐transferrin, | — | — | — | 1994.841 | −17.029 | 22 |
| CACSNHEPYFGYSGAFK (200–216) | 1977.812 | 23 | ||||
| Human serum albumin, | — | — | — | 1552.606 | −17.026 | 14 |
| CCAAADPHECYAK (357–369) | 1535.580 | 15 | ||||
| Ribonuclease A, | 2517.222 | −17.020 | 57 | 2517.218 | −17.018 | 25, 26 |
| CKPVNTFVHESLADVQAVCSQK (39–60) | 2500.202 | 2500.200 | 27 | |||