A composition comprising a eukaryotic cell, which cell comprises a protein, wherein the protein comprises at least one unnatural amino acid comprising a first reactive group, which unnatural amino acid is incorporated into the protein using an orthogonal aminoacyl tRNA synthetase (O-RS) in vivo in the eukaryotic cell, wherein said unnatural amino acid is capable of undergoing at least one post-translational modification, and wherein the at least one post-translational modification comprises attachment of a molecule comprising a second reactive group by a [3+2] cycloaddition to the first reactive group of the at least one unnatural amino acid in vitro outside of the eukaryotic cell, and wherein: (a) the O-RS or a portion thereof is encoded by a polynucleotide sequence as set forth in any one of SEQ ID NO: 20-35; (b) the O-RS comprises an amino acid sequence as set forth in any one of SEQ ID NO: 48-63; or (c) the O-RS comprises an amino acid sequence that is at least 90% identical to that of the naturally occurring tyrosyl aminoacyl-tRNA synthetase (TyrRS) of SEQ ID NO: 2 and comprises the following amino acid substitutions: valine, isoleucine, leucine, glycine, serine, alanine, or threonine at a position corresponding to Tyr37 of SEQ ID NO: 2; and threonine, serine, arginine, asparagine, or glycine at a position corresponding to Asp182 of SEQ ID NO: 2, and optionally further comprises one or more of the following amino acid substitutions: aspartate at a position corresponding to Asn126 of SEQ ID NO: 2; methionine, alanine, valine, or tyrosine at a position corresponding to Phe183 of SEQ ID NO: 2; and serine, methionine, valine, cysteine, threonine, or alanine at a position corresponding to Leu186 of SEQ ID NO: 2. (a) the O-RS or a portion thereof is encoded by a polynucleotide sequence as set forth in any one of SEQ ID NO: 20-35; (b) the O-RS comprises an amino acid sequence as set forth in any one of SEQ ID NO: 48-63; or (c) the O-RS comprises an amino acid sequence that is at least 90% identical to that of the naturally occurring tyrosyl aminoacyl-tRNA synthetase (TyrRS) of SEQ ID NO: 2 and comprises the following amino acid substitutions: valine, isoleucine, leucine, glycine, serine, alanine, or threonine at a position corresponding to Tyr37 of SEQ ID NO: 2; and threonine, serine, arginine, asparagine, or glycine at a position corresponding to Asp182 of SEQ ID NO: 2, and optionally further comprises one or more of the following amino acid substitutions: aspartate at a position corresponding to Asn126 of SEQ ID NO: 2; methionine, alanine, valine, or tyrosine at a position corresponding to Phe183 of SEQ ID NO: 2; and serine, methionine, valine, cysteine, threonine, or alanine at a position corresponding to Leu186 of SEQ ID NO: 2. valine, isoleucine, leucine, glycine, serine, alanine, or threonine at a position corresponding to Tyr37 of SEQ ID NO: 2; and threonine, serine, arginine, asparagine, or glycine at a position corresponding to Asp182 of SEQ ID NO: 2, and optionally further comprises one or more of the following amino acid substitutions: aspartate at a position corresponding to Asn126 of SEQ ID NO: 2; methionine, alanine, valine, or tyrosine at a position corresponding to Phe183 of SEQ ID NO: 2; and serine, methionine, valine, cysteine, threonine, or alanine at a position corresponding to Leu186 of SEQ ID NO: 2. aspartate at a position corresponding to Asn126 of SEQ ID NO: 2; methionine, alanine, valine, or tyrosine at a position corresponding to Phe183 of SEQ ID NO: 2; and serine, methionine, valine, cysteine, threonine, or alanine at a position corresponding to Leu186 of SEQ ID NO: 2.