The study evaluates the capabilities of two-dimensional gel electrophoresis (2DE) combined with mass spectrometry (MS) for proteome analysis. Using a narrow pH range (4.9–5.7) 2D gel, over 1,500 protein spots were visualized from 0.5 mg of yeast protein. Fifty spots were identified by MS, but proteins from genes with codon bias values <0.1 (low-abundance proteins) were not detected, despite these genes comprising more than half of yeast genes. Increasing the protein load to 50 mg and fractionating the proteins further improved the detection of low-abundance proteins. The results suggest that the 2DE-MS approach is limited in characterizing medium to low-abundance proteins, which is a significant challenge for comprehensive proteome analysis. The study highlights the need for novel techniques to handle larger protein loads and enable large-scale quantitative comparisons of protein expression.The study evaluates the capabilities of two-dimensional gel electrophoresis (2DE) combined with mass spectrometry (MS) for proteome analysis. Using a narrow pH range (4.9–5.7) 2D gel, over 1,500 protein spots were visualized from 0.5 mg of yeast protein. Fifty spots were identified by MS, but proteins from genes with codon bias values <0.1 (low-abundance proteins) were not detected, despite these genes comprising more than half of yeast genes. Increasing the protein load to 50 mg and fractionating the proteins further improved the detection of low-abundance proteins. The results suggest that the 2DE-MS approach is limited in characterizing medium to low-abundance proteins, which is a significant challenge for comprehensive proteome analysis. The study highlights the need for novel techniques to handle larger protein loads and enable large-scale quantitative comparisons of protein expression.