Protein identification and peptide sequencing by tandem mass spectrometry requires knowledge of how peptides fragment in the gas phase, specifically which bonds are broken and where the charge(s) resides in the products. For many peptides, cleavage at the amide bonds dominate, producing a series of ions that are designated b and y. For other peptides, enhanced cleavage occurs at just one or two amino acid residues. Surface-induced dissociation, along with gas-phase collision-induced dissociation performed under a variety of conditions, has been used to refine the general 'mobile proton' model and to determine how and why enhanced cleavages occur at aspartic acid residues and protonated histidine residues. Enhanced cleavage at acidic residues occurs when the charge is unavailable to the peptide backbone or the acidic side-chain. The acidic H of the side-chain then serves to initiate cleavage at the amide bond immediately C-terminal to Asp (or Glu), producing an anhydride. In contrast, enhanced cleavage occurs at His when the His side-chain is protonated, turning His into a weak acid that can initiate backbone cleavage by transferring a proton to the backbone. This allows the nucleophilic nitrogen of the His side-chain to attack and form a cyclic structure that is different from the 'typical' backbone cleavage structures.

Gold surfaces modified with C 3 -C 18 -alkanethiols (CH 3 (CH 2 ) X-1 SH; H X SH; x ) 3, 8, 12, 16, 18) and C 16alkanethiols, fluorinated at the outer 1, 2, 4, and 10 methylene positions (CF 3 (CF 2 ) Y-1 (CH 2 ) X SH; F y H x SH; y ) 1, x ) 15; y ) 2, x ) 14; y ) 4, x ) 12; y ) 10, x ) 6) were characterized by He(I) UV-photoelectron spectroscopy (UPS). (Detailed X-ray photoelectron spectroscopic characterization of the partially fluorinated thin films is given in the Supporting Information). Long incubation times of the gold surface with the alkanethiol solutions lead to compact monolayer films for all of the alkanethiols, as indicated by the exponential decrease in emission intensity versus alkyl chain length for both the gold Fermi edge (UPS data), and by a parallel decrease in Au(4f) photoemission intensity using X-ray photoelectron spectroscopy. Changes in the effective work function of these surfaces due to the presence of significant interfacial dipoles are observed (i) as alkyl chain length is increased, and (ii) as the fraction of fluorinated methylene groups is increased in a constant length alkyl chain. Negative shifts of the low kinetic energy photoemission edge with increasing alkyl chain length in the H x SH series are consistent with the presence of a large positive interface dipole. The largest part of this shift (ca. 1.0 eV) appears between the C 3 -and C 8 -alkyl chain lengths. Adding -CF x groups to the outer end of the C 16 -alkyl chain positively shifts the low-kinetic-energy photoemission edge, consistent with the presence of a large negative interface dipole that completely compensates for the positive dipole from the alkyl portion of the chain. Examining C 13 -C 16 alkyl chains fluorinated at only the outer methyl group shows that this negative dipole depends on the orientation of the -CF 3 group (i.e., "odd-even" effects in the effective work function are observed). Comparison of the shifts in gold/SAM vacuum level (changes in effective work function) as a function of the apparent dipole moment of the molecule provides an estimate of the band-edge offsets for these molecules on the gold surface, an estimate of the intrinsic shift in a vacuum level at zero dipole moment of the adsorbate, and an estimate of the intrinsic dipole moment for the gold-thiolate bond.

Despite decades of accumulated knowledge about proteins and their post-translational modifications (PTMs), numerous questions remain regarding their molecular composition and biological function. One of the most fundamental queries is the extent to which the combinations of DNA-, RNA- and PTM-level variations explode the complexity of the human proteome. Here, we outline what we know from current databases and measurement strategies including mass spectrometry-based proteomics. In doing so, we examine prevailing notions about the number of modifications displayed on human proteins and how they combine to generate the protein diversity underlying health and disease. We frame central issues regarding determination of protein-level variation and PTMs, including some paradoxes present in the field today. We use this framework to assess existing data and to ask the question, "How many distinct primary structures of proteins (proteoforms) are created from the 20,300 human genes?" We also explore prospects for improving measurements to better regularize protein-level biology and efficiently associate PTMs to function and phenotype.