In this paper， muscle tissues of pig， cow， chicken and duck were selected as research objects， and different meat proteins were identified and quantified by data dependent acquisition mode of nano liquid chromatography-QE high resolution mass spectrometry， and their application in identification of adulterated beef meatball was investigated. Results showed that gradient elution parameters of nano LC significantly affected separation efficiency of peptides and identification of proteins. A total of 80 554， 33 350， 40 969 and 32 923 unique peptides belonging to 2 715， 889， 659 and 1 143 proteins were identified from pig， cow， chicken and duck， respectively. 17， 15， 19 and 16 of marker peptides in pig， cow， chicken and duck meat， mainly from myoglobin， myosin， albumin， apolipoproteins and hemoglobin， were identified by comparison of basic local alignment search tool (BLAST) with UniProt database. In data-dependent acquisition mode， 32 of marker peptides from four different muscle tissues showed high signal intensities and good reproducibility， which indicated that these maker peptides were very suitable for identification of meat species and relative quantification of meat proteins. Different protein buffers significantly affected content of protein and signal intensity of marker peptides (P < 0.05)， while chromatographic separation gradients showed little effect on identification and signal intensity of marker peptides. Label-free quantitative proteomics showed that contents of primary proteins were significantly different in beef meatballs with different price (P < 0.05)， and 69.2% of beef meatballs contained pork or chicken， while duck meat was not detected. Moreover， pure beef and adulterated beef meatballs were easily distinguished on the basis of two-dimensional orthogonal partial least squares discriminant analysis.