Table 1.

Identification of proteins physically associated with His-Prt1p by mass measurements of tryptic peptides by using MSa

Gene product identifiedMolecular mass (kDa)No. of peptides observedProbability of correct identificationbFrequency (no. observed/no. predicted) of peptides thatc:Human homolog (% identity with yeast protein)d
ApparenteCalculatedfGene productNext closest candidate gene productContain MetDo not contain Met
Tif32p110110.33212.5 × 10−22 14/1418/18eIF3-p170 (29)
Nip1p9393.21611.0 × 10−4 2/214/14eIF3-p110 (31)
Prt1p9088.12813.7 × 10−8 9/919/19eIF3-p116 (31)
eIF55545.32813.1 × 10−5 9/919/19eIF5 (40)
Tif34p3938.82413.7 × 10−8 1/123/23eIF3-p36 (46)
Tif35p3030.51511.4 × 10−6 1/114/14eIF3-p44 (33)
  • a A sample of Superose-6 column fraction 18 from strain LPY201 (∼1 μg) and an equivalent sample from LPY200 (Fig. 1A) were separated by SDS-PAGE, and the gel was stained with copper stain (Bio-Rad). Six protein bands (numbered 1 to 6 from the top) were found exclusively in the LPY201-derived sample, and these were excised and digested in gel with trypsin. The tryptic peptides were eluted, and their masses were determined by MS (see Materials and Methods). Gel slices were removed from the equivalent positions in the LPY200 lane and treated identically. The masses of all tryptic peptides identified for each band in the LPY201 lane which were absent in the corresponding LPY200-derived sample were compared to the calculated tryptic peptide maps of all yeast proteins in the OWL database, using the ProFound program (see Materials and Methods). The proteins in gel bands 1 and 3 to 6 were identified as Tif32p, Prt1p, eIF5, Tif34p, and Tif35p; band 2 was found to contain Prt1p and Nip1p, which are similar in molecular weight. The search program identified Prt1p as the top candidate, with a probability score of 1, and Nip1p as the second candidate, with a much lower probability score. After assigning all of the measured peptides derived from Prt1p, we used the masses of the remaining peptides to search the database again, and Nip1p was identified as the top candidate, with a probability score of 1.0.

  • b Calculated by the ProFound program.

  • c To confirm the identifications listed in column 1, oxidation of methionine residues in the extracted peptide mixtures was carried out. The masses of peptides shifted by 16 Da following oxidation were presumed to contain methionine; those peptides whose masses did not shift following oxidation were presumed to lack a methionine residue. On average, 30% of the tryptic peptides contained one or more Met residues. For a set of 20 measured tryptic peptides, if 5 contain methionine and 15 do not, the probability of an incorrect identification is further decreased by a factor of (0.3)5× (1 − 0.3)15 = 1.1 × 10−5 below that achieved with database searching with the peptide masses alone. Therefore, the chance of false identification for the six polypeptides listed here is negligible.

  • d The percentages of identity between the yeast polypeptides and their presumed homologs in mammalian eIF3 were calculated by using the Bestfit program in the Genetics Computer Group software package (18). Accession numbers (in parentheses) for the mammalian protein sequences are as follows: eIF3-p170 (D50929 ), eIF3-p110 (U91326 ), eIF3-p116 (U62583 ), eIF5 (P55010 ), and eIF3-p36 (S60335 ). The human eIF3-p44 sequence was made available by Hershey (29a).

  • e Based on mobility in SDS-PAGE analysis.

  • f Based on the amino acid sequences predicted from the DNA sequences of the ORFs retrieved from the following GenBank database records (accession numbers in parentheses): Tif32p (P38249 ), Nip1p (P32497 ), Prt1p (P06103 ), yeast eIF5 (P38431 ), Tif34p (P40217 ), and Tif35p (Z74101 ).