PMC

The CID spectra for the y₃₈⁴⁺ ions of different Ub derivatives were acquired by the LTQ mass spectrometer. Major product ions in the spectra are assigned, and those specific to the Ub linkages are highlighted.

The CID spectra of human UbR74 and two GG-modified forms were acquired by high-resolution Orbitrap. The sequence of the Ub fragments and their major dissociation sites are shown, with all potential ubiquitination sites underlined. Major product ions in the MS/MS spectra are assigned. y₅₆⁶⁺, for example, indicates the y₅₆ ion with six positive charges. Main characteristic product ions of the Ub fragments are highlighted in grey.

Folded ubiquitin is digested only at a single R74 site by trypsin, leading to the generation of an almost full-length ubiquitin (UbR74) and linkage-specific GG-tagged UbR74, such as UbR74-1GG-K48 (one GG tag through K48 residue). Listed are the structures of several ubiquitin forms before and after partial trypsin digestion, including monomer (A), dimer (B), homogeneous and heterogeneous trimers (C and D, respectively), and forked trimer (E).

(A) K48-linked Ub dimers were digested into equal molar amount of UbR74 and UbR74-1GG-K48. The co-elution of UbR74 fragment (mass range: 845.7–846.7 m/z) and its GG-tagged form (mass range: 857.1–858.1 m/z) during C₈ reverse phase chromatography. (B) The measured ratio of the two Ub fragments by LC-MS. (C) K63-linked Ub tetramers were trypsinized into UbR74 and UbR74-1GG-K63 with expected 1:3 ratio. (D) The quantification of two Ub fragments based on different charge state, ranging from 7+ to 13+. The expected ratios are shown in dashed lines. When summarizing the ratio for Ub tetramer, the data point of charge state +13 was removed as an outlier due to weak signal.

(A) Recombinant yeast ubiquitin was fused with His and myc tags at the N-terminus (His-myc-yUb). Native free polyUb chains were purified from yeast by Nickel affinity chromatography and glycerol gradient centrifugation, which was analyzed on a silver-stained gel. (B) MS analysis of the His-myc-yUb sample before and after partially tryptic digestion. (C) The deconvoluted mass spectra (1+ charge) of five major Ub ions. (D) The MS/MS spectra of yUbR74-1GG acquired using LTQ in profile mode. Some regions of the spectra were zoomed for the display: 650–850 m/z, 10 fold; 850–1015 m/z, 5 fold; 1015–1145 m/z and 1155–1400 m/z, 2 fold. Linkage-specific product ions were highlighted.

(A) Human ubiquitin monomer was detected as multiple charge states during LC-MS. The most abundant isotopic ions for all charge states (m/z) are indicated. (B) The distribution of isotopic ions with ten positive charges measured by high resolution Orbitrap mass spectrometry. (C) The effect of trypsin/substrate ratio on the digestion efficiency of ubiquitin. Human ubiquitin was incubated with trypsin at 5 μg/ml overnight at 37 oC. Bovine serum albumin was added in to adjust trypsin/substrate ratio as indicated. The digested products were analyzed by LC-MS to detect the UbR74 fragment. (D) The kinetics of ubiquitin digestion. Human ubiquitin was digested with trypsin at 5 μg/ml and enzyme-to-substrate ratio of 1:3. An aliquot was taken at 20 min, 1 hr, 3 hr and 9 hr for mass spectrometric analyses.