Table 1. Mass spectrometric analysis of lysine residues used for polyubiquitin chain formation

Peptide

Sequence

Lysine ubiquit.

Observed mass (M+H)

Expected mass (M+H)

1–6

MQIFK

—

765.69

764.43

7–27

TLTGKTITLEVEPSDTIENVK +GG

K11

2,402.00

2400.99

12–27

TITLEVEPSDTIENVK

—

1,787.74

1,786.73

30–42

IQDKEGIPPDQQR

—

1,523.74

1,522.73

34–42

EGIPPDQQR

—

1,039.63

1,038.62

43–48

LIFAGK

—

648.90

647.40

43–54

LIFAGKQLEDGR +GG

K48

1,460.74

1,459.73

49–54

QLEDGR

—

717.71

716.35

55–63

TLSDYNIQK

—

1,081.64

1,080.63

55–72

TLSDYNIQKESTLHLVLR +GG

K63

2,244.02

2,243.01

55–74

TLSDYNIQKESTLHLVLRLR +GG

K63

2,513.13

2,512.13

64–72

ESTLHLVLR

—

1,067.73

1,066.72

The lysine residues of ubiquitin used for polyubiquitin chain formation in the presence of Hdm2 alone or in the presence of Hdm2 and HdmX were determined by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry in three independent experiments. Polyubiquitinated proteins were digested with trypsin and ubiquitinated peptides identified by a mass increment of +114 Da, corresponding to the two C-terminal glycine residues of ubiquitin (+GG) that remain covalently attached to the respective lysine residue after trypsin digestion. The sequences of the identified peptides (the respective amino acid residues of ubiquitin are indicated) and the observed and the expected mass of the respective peptide are shown. Peptides spanning amino acid residues 28 and 29 were not observed due to their low mass (trypsin cuts behind amino acid residues 27 and 29). Note that, if lysine 29 were to be used for ubiquitin chain formation, trypsin would not cut behind lysine 29 and, thus, peptides of a size readily detectable in mass spectrometry would be generated.