Studying biomolecule deprotonated anions (amino acids, amino acid amides, phosphorylated amino acids, phosphorylated amino acid amides, and phosphorylated peptides) can further negative ion mode mass spectrometry work in the field of proteomics, the sequencing of human proteins. Traditionally, amino acids are considered to have acidic sites on their C-terminus and carboxylic acid-containing side chains. However, several non- traditional sites capable of deprotonation should be considered when examining peptides and proteins by mass spectrometry. A new deprotonated tyrosine conformer was found by electrospraying (ESI) tyrosine from aprotic solvents. Ion/molecule (I/M) reactions determined that tyrosine made by ESI from protic solvents is less acidic. The gas-phase acidity (GA) value for the low-energy structure is 324.7 ± 3.6 kcal/mol experimentally and 330.4 kcal/mol computationally. The ESI conditions and hexapole trapping can affect the deprotonation site of tyrosine. The GAs for the common amino acid amides, phosphorylated amino acids, and phosphorylated amino acid amides were determined experimentally and computationally. Two ion populations were observed via I/M reactions for the amino acid amides deprotonating on the C-terminal amide group that vary in energy by ~5 kcal/mol . Tyrosine, cysteine, tryptophan, and histidine amides undergo side chain deprotonation and are more acidic. Phosphorylated amino acids and their amides were found to be ~20 kcal/mol more acidic than their non-phosphorylated counterparts. Phosphorylated compounds deprotonate on the phosphate side chain, except phosphotyrosine (pTyr), which deprotonates at the C-terminal carboxylic acid. The acidity of pTyr can be affected by ESI conditions. Collison-induced dissociation (CID) was used to examine model phosphorylated peptides in the negative ion mode. The CID of singly charged deprotonated precursor ions produced the least amount of sequence informative fragmentation when the phosphorylated residue was located centrally in the peptide. Diagnostic ions and losses were found indicating the phosphate group. Phosphothreonine (pThr) and phosphoserine (pSer) undergo the loss of the phosphate group and side chain aldehyde to produce unique marker ions. Fragmentation of doubly charged precursor anions yielded little sequence informative fragmentation, however; diagnostic product ions indicating loss of the phosphate group allowed for differentiation between pThr, pSer, and pTyr.