Determination of size, molecular weight, and presence of subunits.

The size or apparent molecular weight of a given protein may be the most cited distinguishing characteristic of the molecule. In addition to being the basis of many separation methods, the molecular weight, or simply molecular size, immediately provides the investigator with an idea of the complexity of the molecule, whether it is likely to be difficult to produce in quantity, and whether certain analytical methods are likely to be productive. Knowing whether the polypeptide of interest can self assemble or exists in a heterogeneous complex with other polypeptides may provide valuable information regarding biosynthesis or mechanism. This chapter outlines key methods used to determine the size of proteins, their molecular weight, and whether subunits are present, with a focus on the basis of the determinations, their strengths, and their limitations.

Baseline resolution of isobaric phosphorylated and sulfated peptides and nucleotides by electrospray ionization FTICR ms: another step toward mass spectrometry-based proteomics.

Electrospray ionization broadband FTICR mass spectrometry at a mass resolving power, m/delta m50% > or = 400,000 has achieved the first direct mass spectral resolution of phosphorylated and sulfated peptides (or nucleotides) of the same nominal mass. The elemental composition difference in each case is PH versus S (9.5 mDa), requiring a minimum mass resolving power ((m2 - m1)/ml) of 118,000 (C terminal amidated cholecystekinin fragment 26-33 (CCK-8), DY(PO3H2)MGWMDF-NH2 versus DY(SO3H)MGWMDF-NH2) or 65,400 (adenosine triphosphate vs 3-phosphoadenosine 5'-phosphosulfate). The isobaric mass doublets were detected in broadband mode (400 < m/z <1400) in the presence of dozens of other species. It is therefore now possible to distinguish phosphorylated from sulfated peptides, even when both species are present at the same time in a protein digest.