Linking genome and proteome by mass spectrometry: large-scale identification of yeast proteins from two dimensional gels

Abstract

The function of many of the uncharacterized open reading frames discovered by genomic sequencing can be determined at the level of expressed gene products, the proteome. However, identifying the cognate gene from minute amounts of protein has been one of the major problems in molecular biology. Using yeast as an example, we demonstrate here that mass spectrometric protein identification is a general solution to this problem given a completely sequenced genome. As a first screen, our strategy uses automated laser desorption ionization mass spectrometry of the peptide mixtures produced by in-gel tryptic digestion of a protein. Up to 90% of proteins are identified by searching sequence data bases by lists of peptide masses obtained with high accuracy. The remaining proteins are identified by partially sequencing several peptides of the unseparated mixture by nanoelectrospray tandem mass spectrometry followed by data base searching with multiple peptide sequence tags. In blind trials, the method led to unambiguous identification in all cases. In the largest individual protein identification project to date, a total of 150 gel spots-many of them at subpicomole amounts-were successfully analyzed, greatly enlarging a yeast two-dimensional gel data base. More than 32 proteins were novel and matched to previously uncharacterized open reading frames in the yeast genome. This study establishes that mass spectrometry provides the required throughput, the certainty of identification, and the general applicability to serve as the method of choice to connect genome and proteome.

Figure 1

A strategy based exclusively on mass spectrometry to identify proteins separated by 2D PAGE with certainty, sensitivity, and high throughput.

Figure 2

Identification of yeast protein ILV5 by automated MALDI mass spectrometry and automated data base searching. Ion signals whose measured masses match calculated masses of protonated tryptic peptides, (M + H)⁺, within 50 ppm are indicated with circles. Terminal sequence tags (35) are marked by arrows and with the amino acid producing the ragged end pattern. (Inset) A magnification of one of the tryptic peptide peaks showing isotopically resolved signals differing by 1 Da due to the natural ¹³C abundance. Sequence coverage is greater than 70 percent. Average absolute mass accuracy is 25 ppm and average resolution is 10,000. Some of the unmarked peaks are matrix related, some are trypsin autolysis products and two matching peptides are outside the mass range shown.

Figure 3

High sensitivity protein identification by nano electrospray mass spectrometry. (A) Mass spectrum (Q₁ scan) of a tryptic digest of a spot marked by an arrow on Fig. 4. Ions of trypsin autolysis products are marked (∗). (B) Parent ion scan spectrum for the immonium ions of Leu/Ile (m/z 86) acquired using the same digest. Ions of tryptic peptides are designated T_A–T_C. (C) Tandem mass spectrum of the doubly charged ion T_B²⁺. The first mass of the fragmentation series, the sequence as determined by the mass differences between fragment peaks and the last mass in the series were entered as (600.2)ED(L/I)(957.4) into peptidesearch together with the measured peptide mass. Yeast protein VMA2 was unambiguously identified by the peptide sequence tag as shown in the spectrum. The letters refer to N-terminal (a and b) and C terminal (y) fragmentation at the amide bonds (38).

Figure 4

2D PAGE yeast reference map with protein identifications as determined in the current investigation. The protein pattern corresponds to [³⁵S]methionine-labeled polypeptide (19). The arrow indicates the protein sequenced in Fig. 3 and the rectangles indicate proteins of the yeast L-A virus. The elipses mark locations of very weakly staining proteins. Gene names of previously uncharacterized open reading frames are in boldface type.