Figure 5. Positive mode MALDI-TOF-MS of MDR i (Control) and its 15 N, 13 C-labeled counterpart showed the [M+H] + ions for the tryptic peptides. Fragments at m/z 1041.6 (top) and 1103.7 (bottom) were unexpectedly observed; thus, the former was subjected to tandem MS (see Fig. 7). Meanwhile, isotope patterns for the 15 N, 13 C-MDR i indicate random underlabeling. OVERVIEW Stability Evaluation of HIV Type 1 Protease Constructs by High-Resolution Mass Spectrometry Ian Mitchelle S. De Vera 1 , Gail E. Fanucci 1 , and Maria Cristina A. Dancel 1,2 1 Department of Chemistry, University of Florida, Gainesville, FL 32611 2 Mass Spectrometry Facility, University of Florida, Gainesville, FL 32611 HIV-1PR is a 99-amino acid homodimeric aspartic protease that processes gag and gag-pol polypeptides. It is an attractive target for AIDS antiviral therapy because of its central role in viral maturation [1]. Protease inhibitors (PIs) that specifically target HIV-1PR prevent the formation of infectious virions and interfere with viral maturation. The efficacy of currently available PIs is limited by the rapid emergence of resistance mutations in this protease. These mutations include the active site mutation D30N that occurs only in patients exposed to nelfinavir (NFV) [2], and secondary mutations M36I and A71V that contribute to ritonavir (RTV) and indinavir (IDV) cross- resistance [3]. Figure 1 shows the active (D25) subtype B construct, B sa which will be used in enzyme kinetics assays and the multi-drug resistant construct MDR i , which was isotopically-labeled for triple resonance (3D) NMR experiments. To retard the autoproteolysis of B sa , the inactivating mutation (D25N) is incorporated to generate the construct, B si . HIV-1PR B si constructs that possess drug-resistance associated mutations are spin-labeled at the K55C and K55C’ reporter sites (Figure 2), rendering the protease amenable to DEER measurements. Site-directed mutagenesis was performed on a B sa template to generate seven inactive (D25N) constructs: D30N, M36I, A71V, D30N/M36I, D30N/A71V, M36I/A71V, and D30N/M36I/A71V. Fidelity of the HIV-1PR genes were confirmed by Sanger DNA sequencing. B si constructs with engineered cysteine residues (K55C), MDR i , and B sa were expressed under the control of a T7 promoter into inclusion bodies using BL21*(DE3) pLysS E. coli cells. Site-directed spin labeling was accomplished by incubating the protease with three-fold molar excess of (1-oxyl-2,2,5,5-tetramethyl-∆3-pyrroline-3-methyl) methanethiosulfonate (MTSL) for 12 hours. The protein was reconstituted in 2 mM NaOAc and concentrated to OD 280 > 1.0 prior to DEER measurements and electrospray ionization (ESI) with Agilent 6210 TOF-MS. Some of these constructs were re-analyzed after several months to check the stability of MTSL label. In addition, MDR i was expressed in M9 media with 15 NH 4 Cl and 13 C-glucose for uniform isotopic labeling. The protease was reconstituted in 20 mM deuterated NaOAc and concentrated to OD 280 of at least 2.0 before collecting triple resonance (3D) NMR and ESI-TOF-MS. In-solution trypsin digestion was done in a 40 o C water bath for three hours. Aliquots were collected every hour for B sa . Control samples were heated without the enzyme to determine thermal degradation products. The peptides were mixed with α-cyano-4-hydroxycinnamic acid (CHCA) and trifluoroacetic acid (TFA), spotted on a plate, and analyzed using an AB Sciex TOF/TOF 5800 instrument in positive (+) and negative (-) modes. Ions of interest underwent tandem MS and de novo sequencing.  MDR i Trypsin Digests METHODOLOGY PRELIMINARY RESULTS  ESI-TOF-MS and MALDI-TOF-MS of HIV-1 Protease Constructs Table 1. Molecular weights of intact protease via ESI-TOF-MS and percent amino acid coverage of trypsin digests via MALDI-TOF-MS ACKNOWLEDGMENTS • NSF CRIF Grant 0541761 and CHE MRI 1040016 for the mass spectrometers • NIH AIDS Research and Reference Reagent Program • NSF Career Award and NHMFL IHRP • University of Florida Center for AIDS Research • University of Florida Startup Funds REFERENCES [1] Huff, J.R., J. Med. Chem., 1991, 34, 2305-2314. [2] Weber, I.T.; Agniswamy, J. Viruses, 2009, 1, 1110-1136. [3] Kantor, R. et al. Ant. Agents and Chemoth., 2007, 46, 1086-1092. [4] Mildner, A.M. et al. Biochemistry, 1994, 33, 9405-9413. [5] Perona, J.J.; Craik, C.S., Protein Sci., 1995, 4, 337-360. HIV-1 PR MTSL HIV-1 PR-MTSL Table 1 shows that ESI-TOF-MS gives average molecular weights within +0.2 Da of theoretical values; thus, intact proteins that are 1-Da apart are easily differentiated. Meanwhile, MALDI-TOF-MS in both positive and negative mode increased AA coverage of trypsin-digested HIV-1PR constructs to > 98%. ND = not digested; # = total coverage based on both positive and negative modes Figure 3. Deconvoluted ESI-TOF-MS spectrum of the triple mutant, after eight months of storage at -20 o C, shows disulfide-linked dimer formation after the MTSL fell off. The nitroxide label was intact for all other protease constructs even after three months at 4 o C.  B sa Autoproteolysis and Thermal Degradation Analysis Figure 6. Negative mode MALDI-TOF-MS of MDR i (Control) and its 15 N, 13 C-labeled counterpart showed [M-H] - ions for the trypsin digests. A few peptides, i.e., m/z 958.6 & 1495.7 (top) and the corresponding m/z 1015.6 & 1580.8 (bottom), were only detected in negative mode. Figure 4. MALDI-TOF-MS of B sa , after storage at -20 o C(Control) and after heating at 40 o C for an hour, show some laser ablation fragments and the expected autolysis fragments at m/z 3449 and 3713 [4]. Ions below m/z 2000 after 2 hours or more indicate thermal degradation products.  Stability evaluation of MTSL labeling by ESI-TOF-MS CONCLUSIONS INTRODUCTION Our goal is to determine the stability of various HIV type 1 protease (HIV-1PR) constructs that were used in triple resonance nuclear magnetic resonance (3D NMR) spectroscopy experiments and conformational studies via double electron-electron resonance (DEER) spectroscopy. Intact proteins underwent electrospray ionization (ESI) while trypsin digests were subjected to matrix-assisted laser desorption ionization (MALDI), followed by time-of-flight mass spectrometry (TOF-MS). Results show molecular weights with less than 0.2-Da mass error; meanwhile, trypsin digests confirmed the amino acid sequences of the different constructs.  ESI-TOF-MS results reveal that the MTSL-labeled constructs are stable at -20 o C, except for B si D30N/M36I/A71V that formed the disulfide-linked dimer after 8 months.  B sa is detectable even after several days at room temperature based on ESI-TOF-MS (data not shown), but MALDI-TOF-MS indicates that it undergoes autoproteolysis to some degree even at -20 o C and thermally degrades after 2 or more hours at 40 o C.  Percent AA coverage of trypsin digests improved by collecting MALDI-TOF-MS spectra in positive and negative modes.  Incomplete isotope-labeling of MDR i was random and not residue-specific. Figure 7. A tryptic peptide at m/z 1041.6 was observed in the MALDI-TOF(+)-MS spectrum of MDR i and was subjected to tandem MS (shown above). The de novo sequence search identified isobaric peptides of PQITLWQR (see inset). This fragment comes from enzymatic cleavage between R & P that usually occurs at a much slower rate [5], and was not observed in other HIV-1PR constructs. MTSL‐labeled Disulfide‐linked Dimer n M/n M n X/n X 6 1,815.95 10,895.70 13 1,647.78 21,421.09 7 1,556.53 10,895.72 14 1,530.08 21,421.15 8 1,361.97 10,895.76 15 1,428.08 21,421.18 9 1,210.65 10,895.86 16 1,338.83 21,421.31 10 1,089.60 10,895.97 17 1,260.07 21,421.22 11 990.55 10,896.01 18 1,190.07 21,421.30 12 908.00 10,895.96 19 1,127.44 21,421.31 13 838.14 10,895.86 20 1,071.07 21,421.31 14 778.28 10,895.89 21 1,020.06 21,421.21 15 726.40 10,896.00 Average 21,421.23 Average 10,895.87 SD 0.08 SD 0.12 [(M+H)-H2O]+ b1 b2 b3 b4 b5 b6 b7 y1 y2 y3 489.4 y4 y5 y6 y7 [M+H]+ 1041.6 [(M+H)-NH3]+ [(M+H)-44]+ Construct MW by ESI-TOF-MS % AA Coverage by MALDI-TOF-MS Theoretical Mass Observed Mass Positive Mode Negative Mode Total # Bsi M36I K55C +MTSL 10868.90 10868.72 ±0.11 88 79 100 Bsi D30N M36I K55C +MTSL 10867.91 10867.81 ±0.10 88 73 100 Bsi K55C +MTSL 10886.95 10887.00 ±0.03 ND ND ND Bsi D30N K55C +MTSL 10885.96 10885.92 ±0.12 88 73 100 Bsi M36I A71V K55C +MTSL 10896.94 10897.05 ±0.05 ND ND ND Bsi D30N M36I A71V K55C +MTSL 10895.96 10895.91 ±0.11 88 73 100 Bsi A71V K55C +MTSL 10914.98 10915.00 ±0.12 ND ND ND Bsi D30N A71V K55C +MTSL 10914.00 10914.03 ±0.15 88 73 100 Bsa 10728.69 10728.68 ±0.07 86 81 98 MDRi 10637.47 10637.50 ±0.09 86 65 98 MDRi 15 N, 13 C-labeled 11248.91 11242.46 ±0.15 86 65 98 Figure 1. Amino acid sequence of B sa and MDR i . Yellow residues correspond to the inactivating mutation D25N. Residues in red are mutations in MDR i with respect to subtype B. 10 20 30 40 50 B sa PQITLWKRPL VTIKIGGQLK EALLDTGADD TVIEEMSLPG RWKPKMIGGI MDR i PQITLWQRPI VTIKIGGQLK EALLNTGADD TVLEEVNLPG RWKPKLIGGI 60 70 80 90 B sa GGFIKVRQYD QIIIEIAGHK AIGTVLVGPT PVNIIGRNLL TQIGATLNF MDR i GGFVKVRQYD QVPIEIAGHK VIGTVLVGPT PANVIGRNLM TQIGATLNF Figure 2. Ribbon diagram of HIV-1PR (left) rendered using PyMol, showing the active site (D25) and spin-labeling sites (K55C and K55C’). Mutation sites are shown as spheres, with active site mutation D30N shown in cyan and secondary M36I and A71V mutations shown in pink and yellow, respectively. The sulfhydryl-specific reaction (right) yielding the disulfide-linked spin label.