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Post-translational modification of proteins by Ubiquitin (Ub) and Ubiquitin-like proteins (Ubls) can be reversed by deconjugating enzymes, which have been implicated in different pathways and associated with various human diseases. To understand the activity and dynamics of deconjugating enzymes, multiple synthetic and semi-synthetic Ub/Ubl probes have been developed, and some of them have been applied to screen inhibitors of deconjugating enzymes. Since these Ub/Ubl probes are generally not cell-permeable, different strategies have been developed to deliver Ub/Ubl probes to live cells. However, till now, no Ub/Ubl probes can be expressed in live cells to directly report on the activities of deconjugating enzymes in the most relevant cellular environment. Here, we genetically encoded cross-linkable Ub/Ubl probes in live

A key step in the workflow of bottom-up proteomics is the proteolysis of proteins into peptides with trypsin. In addition, enzyme-catalytic (18)O labeled peptides as internal standards coupled with multiple reaction monitoring mass spectrometry (MRM MS) for the absolute quantitation of the target proteome is commonly used for its convenient operation and low cost. However, long digestion and labeling times, incomplete digestion and (18)O to (16)O back exchange limit its application, therefore, we developed a rapid and efficient digestion method based on a high ratio of trypsin to protein. In addition, after separation of the digested samples using pipette tips packed with reversed-phase packing materials in house, the trypsin can be separated, collected and reused at least four times. Based on this approach, a novel protein quantification method using (18)O-labeled QconCAT peptides as internal standards combined with MRM MS for the absolute quantitation of a target proteome is established. Experimental results showed that the novel method had high digestion and (18)O labeling efficiencies, and no (18)O to (16)O back-exchange occurred. A linear range covering 2 orders of magnitude and a limit of quantification (LOQ) as low as 5 fmol were achieved with an RSD below 10%. Then, the quantitative method is used for the absolute quantitation of drug metabolizing enzymes in human liver microsomes. The results are in good agreement with the previously reported data, which demonstrates that the novel method can be used for absolute quantitative analyses of target proteomes in complex biological samples.

We applied immobilized enzyme reactors prepared with different sizes of magnetic particles into protein and proteome digestion. In addition, the influences of different sizes of the magnetic particles were studied on the reunion, enzyme efficiency and leakage sites. The experimental results showed that in comparison with the submicron magnetic particles, the amount of trypsin immobilized on the magnetic nanoparticles was 3. 5 times more than that of the submicron magnetic particles. However, the enzymatic efficiency was at the same level when the same amount of trypsin was used, and the reunion phenomenon was obviously improved when the size of the magnetic nanoparticles increased. Taking the immobilized enzyme reactor of 20 nm magnetic nanoparticles as an example, the digestion performance was further examined. The experimental results showed that rapid digestion could be achieved within 1 mm when the mass ratio of the trypsin and bovine serum albumin was 1:1. The peptide number of 0 missed cleavage site and the sequence coverage changed little after the protein was digested for 10 mm. It was concluded that the digestion efficiency of the immobilized enzyme reactor was much better than that of the in-solution digestion. When the immobilized enzyme reactors and the free trypsin were used for digestion, little differences of the leakage sites were found. Therefore, the immobilized enzyme reactors prepared with different sizes of magnetic particles can be applied in proteomic research for quick and efficient digestion.

A method of liquid chromatographic retention time-dependent intelligent selected reaction monitoring mass spectrometry (iSRM MS) was established for the identification of targeted proteome, and was compared in detail with the method without liquid chromatographic retention time-dependent iSRM MS in the analysis results of different samples, such as the peptide mixtures from bovine serum albumin, six standard proteins or Thermoanaerobacter tengcongensis extract digested by trypsin. The results showed that the throughput of the identified peptides and the proteins was evidently increased with the method of liquid chromatographic retention time-dependent iSRM MS, especially in the complex sample such as Thermoanaerobacter tengcongensis extract. The coverage of the identified peptides and proteins from Thermoanaerobacter tengcongensis extract was reached 89.62% and 92.41% of the number of targeted peptides and proteins respectively. Higher sensitivity and reproducibility were also obtained. In addition, peptides with the same m/z but different retention times could be identified accurately when the method was used. With the higher throughput, better sensitivity and excellent reproducibility, the method of liquid chromatographic retention time-dependent iSRM MS can play an important role in the identification of targeted proteomes of complex biological samples in the future.

A method of concatamers of Q peptides (QconCATs) protein labeled with 18O-multiple reaction monitoring mass spectrometry for absolute quantification of proteins is established. The purity of the QconCAT recombinant protein was characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and its purity was above 99%. The relative molecular mass was approximately 63.4 kDa. The peptides digested from the QconCAT recombinant protein and the extract of Thermoanaerobacter tengcongensis (TTE) were analyzed by mass spectrometry. The raw data were processed by pFind and pLabel softwares. The results showed that the efficiencies of protein digestion and the 18O labeling efficiency were able to meet the need of the protein quantification. The performance of the method was evaluated. The absolute contents of the selected proteins in TTE were determined with the relative standard deviations of less than 20% and the accuracy is high. The method not only avoid using the expensive reagent of stable isotope labeling with amino acids in cell culture (SILAC), but also provides an alternative way for the accurately absolute quantification of proteins in biological samples for quantitative proteomic research.

We applied immobilized enzyme reactors prepared with different sizes of magnetic particles into protein and proteome digestion. In addition, the influences of different sizes of the magnetic particles were studied on the reunion, enzyme efficiency and leakage sites. The experimental results showed that in comparison with the submicron magnetic particles, the amount of trypsin immobilized on the magnetic nanoparticles was 3. 5 times more than that of the submicron magnetic particles. However, the enzymatic efficiency was at the same level when the same amount of trypsin was used, and the reunion phenomenon was obviously improved when the size of the magnetic nanoparticles increased. Taking the immobilized enzyme reactor of 20 nm magnetic nanoparticles as an example, the digestion performance was further examined. The experimental results showed that rapid digestion could be achieved within 1 mm when the mass ratio of the trypsin and bovine serum albumin was 1:1. The peptide number of 0 missed cleavage site and the sequence coverage changed little after the protein was digested for 10 mm. It was concluded that the digestion efficiency of the immobilized enzyme reactor was much better than that of the in-solution digestion. When the immobilized enzyme reactors and the free trypsin were used for digestion, little differences of the leakage sites were found. Therefore, the immobilized enzyme reactors prepared with different sizes of magnetic particles can be applied in proteomic research for quick and efficient digestion.

A method of liquid chromatographic retention time-dependent intelligent selected reaction monitoring mass spectrometry (iSRM MS) was established for the identification of targeted proteome, and was compared in detail with the method without liquid chromatographic retention time-dependent iSRM MS in the analysis results of different samples, such as the peptide mixtures from bovine serum albumin, six standard proteins or Thermoanaerobacter tengcongensis extract digested by trypsin. The results showed that the throughput of the identified peptides and the proteins was evidently increased with the method of liquid chromatographic retention time-dependent iSRM MS, especially in the complex sample such as Thermoanaerobacter tengcongensis extract. The coverage of the identified peptides and proteins from Thermoanaerobacter tengcongensis extract was reached 89.62% and 92.41% of the number of targeted peptides and proteins respectively. Higher sensitivity and reproducibility were also obtained. In addition, peptides with the same m/z but different retention times could be identified accurately when the method was used. With the higher throughput, better sensitivity and excellent reproducibility, the method of liquid chromatographic retention time-dependent iSRM MS can play an important role in the identification of targeted proteomes of complex biological samples in the future.

A novel method has been established based on metal element chelated tags coupled with high performance liquid chromatography-selected ion monitoring mass spectrometry (HPLC-SIM/MS). The labeling efficiency and stability of metal element chelated tags, the chromatographic retention behavior and MS behavior of the labeled peptides, the linear range and accuracy of this method were examined. The results showed that the metal element chelated tag method has high labeling efficiency and high labeling stability, and the labeled peptides with different kinds of metal tags have consistent chromatographic retention behavior. The method of metal tags coupled with HPLC-SIM/MS has high sensitivity with the limit of quantification (LOQ) up to 1 fmol. The linear range for the method was between 1 fmol to 500 fmol with R2 > 0.99, which means the method has a good linearity. Moreover, this method had an average recovery of 117.01%. The method was used in the absolute quantitation of a protein enolase in Thermoanaerobacter tengcongensis (TTE) with a relative standard deviation of 5.74%, which means high precision. All the results showed that this method is accurate and reliable for the absolute quantitation of proteins. This gives us an alternative for the quantitative determination of proteins in relatively simple biological samples.

The routine proteolysis of proteins is performed in solution, but it suffers from drawbacks such as long incubation time, enzyme autodigestion, and non-reusability. Therefore we here demonstrated that trypsin could be immobilized on silver wire modified by atom transfer radical polymerization to prepare a new kind of enzyme immobilized reactor. The digestion efficiency, repeatability and recovery of the silver wire-trypsin reactor (SW-Trypsin) were evaluated by mass spectrometry (MS) analysis. Highly efficient digestion was achieved by using SW-Trypsin within only 20 min. Standard protein could be almost completely digested with sequence coverage up to 93%, which is higher than that of 79% sequence coverage obtained by in-solution digestion for 16 h. Bovine serum albumin (BSA) was digested eight times within a month by using the SW-Trypsin. The results of sequence coverage were between 89% to 97%, with an average sequence coverage of 94%, which showed that SW-Trypsin had good stability. In addition, the recovery test by using myoglobin (MYO) showed that the recovery rate was 87.67%. At last, the extract from Tengchong thermophilic bacteria was digested by SW-Trypsin in 20 min and in-solution trypsin in 16 h. The results of sequence coverages and the number of identified proteins were similar. Moreover, the ratio of the number of peptides with zero missed cleavages to the number of all identified peptides by using SW-Trypsin was higher than that by in-solution digestion. Also, the SW-Trypsin was easily removed from the digestion solution. Good performances of SW-Trypsin implied that it has a good prospect in the application in future proteomics research.