Yersinia enterocolitica expresses the Ysc type III secretion system (TTSS) for delivery of effector proteins (Yops) to host cells to manipulate immune system of the host. The study on the physiological change of Y. enterocolitica is important in determining how cells efficiently support the expression of virulence factors and adapt to the inside of hosts, which is completely different from the outside environment. Ysc TTSS also secretes Yops into medium when cells are grown in calcium-deficient medium at $37^{circ}C$. In the present work, a phosphoprotein purification kit was used to selectively enrich proteins whose characteristics changed under the Yop production conditions. Phosphoenol pyruvate synthase (PpsA), elongation factor Tu (TufA), and glutaminase were identified by matrix-assisted laser desorption/ionization time-of-flight. These results showed that the affinity to column and/or the steady-state level of PpsA, TufA, and glutaminase are altered under the Yop secretion conditions and suggested that any protein characteristic of these three proteins was changed under the conditions for the virulence factor expression suggested that certain characteristics of these three proteins were changed. This work demonstrated that specific protein purification method can be used to identify proteins from a complex protein mixture of cell extracts with emphasis on the specific characteristics of proteins.